Improved Signal Processing for Mass Shifting Ions in Charge Detection Mass Spectrometry.

The quality of data in charge detection mass spectrometry depends on accurate determination of ion charge. While the method of selective temporal overview of resonant ions (STORI) has proven to be highly enabling for determining the charge of ions that survive for variable amounts of time, it assumes that the ion frequency exactly matches the frequency being used in the calculation. Any mismatches result in low charge estimates. To address this, the misSTORI method was developed to correct these discrepancies. This can significantly reduce the charge measurement errors for samples with unstable masses. As an example, the misSTORI approach can eliminate a 5.7% charge determination error for a VP3-only AAV capsid that shifts 25 ppm in mass.

Determining Collisional Cross Sections from Ion Decay with Individual Ion Mass Spectrometry.

Collision cross section (CCS) measurements determined by ion mobility spectrometry (IMS) provide useful information about gas-phase protein structure that is complementary to mass analysis. Methods for determining CCS without a dedicated IMS system have been developed for Fourier transform mass spectrometry (FT-MS) platforms by measuring the signal decay during detection. Individual ion mass spectrometry (I2MS) provides charge detection and measures ion lifetimes across the length of an FT-MS detection event. By tracking lifetimes for entire ion populations, we demonstrate simultaneous determination of charge, mass, and CCS for proteins and complexes ranging from ∼8 to ∼232 kDa.

Skeletal muscle coverage in undergraduate courses: a comparison of stand-alone human anatomy and anatomy and physiology (A&P) courses.

Anecdotal data suggest great variation in breadth and depth of skeletal muscle coverage between undergraduate human anatomy (HA) and anatomy and physiology (A&P) courses. This manuscript is the second in a series (Reynolds A, Goodwin M, O'Loughlin VD. Adv Physiol Educ 46: 309-318, 2022. doi:https://doi.org/10.1152/advan.00084.2021) that methodically assesses skeletal muscle content coverage across undergraduate HA and A&P courses. The authors developed an online skeletal muscle coverage survey and collected 342 responses worldwide, 156 from instructors of HA courses and 186 from A&P course instructors. Demographic results showed that HA courses are primarily taught at 4-year baccalaureate institutions, have relatively larger class sizes, and are more likely to use human (vs. animal) prosections or dissections. In contrast, A&P courses are primarily taught at community colleges, have relatively smaller class sizes, and are more likely to use animal (vs. human) dissections. HA courses tend to require their students to learn all skeletal muscle aspects (i.e., identification, action, attachments, innervation), whereas A&P courses tend to focus on muscle identification or action only. The proportions of courses that require identification of large, superficial skeletal muscles are similar between the two course types. However, HA courses are more likely to require their students to identify deeper and smaller muscles, including more distal appendicular muscles and pelvic muscles. These differences likely are due, in part, to the more anatomical focus of HA courses and the slightly different student populations between these courses. These findings provide much-needed information about muscular system coverage between HA and A&P courses and may guide instructor discussions about curricula.

General trends in skeletal muscle coverage in undergraduate human anatomy and anatomy and physiology courses.

There is a widely variable breadth of coverage of skeletal muscle content across both undergraduate human anatomy and undergraduate anatomy and physiology (A&P) courses. In response to the need for a more global understanding of the content taught in undergraduate anatomy courses, we developed an online survey (administered through Qualtrics) where both human anatomy and A&P faculty could report skeletal muscle coverage in their courses. The survey also collected comparative demographic institutional data such as the type of institution (community college vs. 4 year), course format, and geographic location of the undergraduate institution. Skeletal muscles surveyed included those listed and described in a typical undergraduate human anatomy text (McKinley MP, O'Loughlin VD, Pennefather O. Human Anatomy (5th ed.), 2017, p. 960). The data indicated some interesting instructional trends regarding muscular system coverage. First, both the "identification" and "action" of specific muscles are taught at a higher frequency than the teaching of either "attachments or innervation." Innervation of specific skeletal muscles is the least taught concept. In each body region, certain muscles were taught with higher frequency than others. This research shows there is a global trend in teaching identification of specific skeletal muscles within each body region and often this is accompanied by teaching actions of said muscles. These general instructional trends may increase our understanding of the anatomical and physiological education our undergraduate students are receiving and will lead to further critical conversations about content development and curriculum.

Novel Real-Time Library Search Driven Data Acquisition Strategy for Identification and Characterization of Metabolites.

Structural characterization of novel metabolites in drug discovery or metabolomics is one of the most challenging tasks. Multilevel fragmentation (MSn) based approaches combined with various dissociation modes are frequently utilized for facilitating structure assignment of unknown compounds. As each of the MS precursors undergoes MSn, the instrument cycle time can limit the total number of precursors analyzed in a single LC run for complex samples. This necessitates splitting data acquisition into several analyses to target lower concentration analytes in successive experiments. Here we present a new LC/MS data acquisition strategy, termed Met-IQ, where the decision to perform an MSn acquisition is automatically made in real time based on the similarity between the experimental MS2 spectrum and a spectrum in a reference spectral library for the known compounds of interest. If similarity to a spectrum in the library is found, the instrument performs a decision-dependent event, such as an MS3 spectrum. Compared to an intensity-based, data-dependent MSn experiment, only a limited number of MS3 are triggered using Met-IQ, increasing the overall MS2 instrument sampling rate. We applied this strategy to an Amprenavir sample incubated with human liver microsomes. The number of MS2 spectra increased 2-fold compared to a data dependent experiment where MS3 was triggered for each precursor, resulting in identification of 14-34% more unique potential metabolites. Furthermore, the MS2 fragments were selected to focus likely sources of useful structural information, specifically higher mass fragments to maximize acquisition of MS3 data relevant for structure assignment. The described Met-IQ strategy is not limited to metabolism experiments and can be applied to analytical samples where the detection of unknown compounds structurally related to a known compound(s) is sought.

A highly conserved host lipase deacylates oxidized phospholipids and ameliorates acute lung injury in mice.

Oxidized phospholipids have diverse biological activities, many of which can be pathological, yet how they are inactivated in vivo is not fully understood. Here, we present evidence that a highly conserved host lipase, acyloxyacyl hydrolase (AOAH), can play a significant role in reducing the pro-inflammatory activities of two prominent products of phospholipid oxidation, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. AOAH removed the sn-2 and sn-1 acyl chains from both lipids and reduced their ability to induce macrophage inflammasome activation and cell death in vitro and acute lung injury in mice. In addition to transforming Gram-negative bacterial lipopolysaccharide from stimulus to inhibitor, its most studied activity, AOAH can inactivate these important danger-associated molecular pattern molecules and reduce tissue inflammation and injury.

Resistance of Mycobacterium tuberculosis to indole 4-carboxamides occurs through alterations in drug metabolism and tryptophan biosynthesis.

Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.

Structure-Activity Relationships of Pyrazolo[1,5-a]pyrimidin-7(4H)-ones as Antitubercular Agents.

Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.

Dynamic Volumetric Assessment of the Aortic Root: The Influence of Bicuspid Aortic Valve Competence.

BACKGROUND: Aortic root evaluation is conventionally based on 2-dimensional measurements at a single phase of the cardiac cycle. This work presents an image analysis method for assessing dynamic 3-dimensional changes in the aortic root of minimally calcified bicuspid aortic valves (BAVs) with and without moderate to severe aortic regurgitation. METHODS: The aortic root was segmented over the full cardiac cycle in 3-dimensional transesophageal echocardiographic images acquired from 19 patients with minimally calcified BAVs and from 16 patients with physiologically normal tricuspid aortic valves (TAVs). The size and dynamics of the aortic root were assessed using the following image-derived measurements: absolute mean root volume and mean area at the level of the ventriculoaortic junction, sinuses of Valsalva, and sinotubular junction, as well as normalized root volume change and normalized area change of the ventriculoaortic junction, sinuses of Valsalva, and sinotubular junction over the cardiac cycle. RESULTS: Normalized volume change over the cardiac cycle was significantly greater in BAV roots with moderate to severe regurgitation than in normal TAV roots and in BAV roots with no or mild regurgitation. Aortic root dynamics were most significantly different at the mid-level of the sinuses of Valsalva in BAVs with moderate to severe regurgitation than in competent TAVs and BAVs. CONCLUSIONS: Echocardiographic reconstruction of the aortic root demonstrates significant differences in dynamics of BAV roots with moderate to severe regurgitation relative to physiologically normal TAVs and competent BAVs. This finding may have implications for risk of future dilatation, dissection, or rupture, which warrant further investigation.

Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins.

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.

Voltage Rollercoaster Filtering of Low-Mass Contaminants During Native Protein Analysis.

Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 µM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.

Inhibition of CorA-Dependent Magnesium Homeostasis Is Cidal in Mycobacterium tuberculosis.

Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

Socioeconomic disparities in health-related quality of life among colorectal cancer survivors.

PURPOSE: Improvements in colorectal cancer (CRC) prevention, early detection, and treatment have resulted in substantial gains in survival. However, the health-related quality of life (HRQoL) of CRC survivors often depends on access to supportive care, which differs by survivors' socioeconomic characteristics. The purpose of this study was to investigate the relationship between socioeconomic characteristics and HRQoL in a diverse group of CRC survivors. METHODS: We conducted a population-based, cross-sectional study to examine the association between socioeconomic factors (household income, health literacy, and insurance status) and HRQoL domains of pain interference, fatigue, physical function, sleep disturbance, anxiety, and depression. PROMIS® Short Forms v.2.0 were used to assess domains of HRQoL. Linear regression modeling was used to estimate the coefficient representing the average HRQoL domain score and its 95% confidence interval (CI). RESULTS: Three hundred one CRC survivors participated in the survey. Low-income (≤ $30,000) CRC survivors had, on average, a 4.70-point (95% CI 1.10-8.28) higher pain interference score, a 7.02-point (95% CI 3.27-10.77) higher fatigue score, a 5.13-point (95% CI - 8.56 to - 1.71) lower physical function score, and a 4.44-point (95% 1.40-7.49) higher depression score than CRC survivors with an income ≥ $70,000. Survivors with Medicaid insurance reported significantly greater pain interference and worse physical function than privately insured survivors. Survivors with low health literacy reported significantly greater pain interference compared with survivors with high health literacy. CONCLUSIONS: Substantial socioeconomic disparities in HRQoL were observed in this diverse population of CRC survivors. IMPLICATIONS FOR CANCER SURVIVORS: Designing supportive care interventions to improve HRQoL among low-income and Medicaid-insured CRC survivors is critical for eliminating disparities in CRC outcomes.

Breaking with Tradition: A Scoping Meta-Analysis Analyzing the Effects of Student-Centered Learning and Computer-Aided Instruction on Student Performance in Anatomy.

While prior meta-analyses in anatomy education have explored the effects of laboratory pedagogies and histology media on learner performance, the effects of student-centered learning (SCL) and computer-aided instruction (CAI) have not been broadly evaluated. This research sought to answer the question, "How effective are student-centered pedagogies and CAI at increasing student knowledge gains in anatomy compared to traditional didactic approaches?" Relevant studies published within the past 51 years were searched using five databases. Predetermined eligibility criteria were applied to the screening of titles and abstracts to discern their appropriateness for study inclusion. A summary effect size was estimated to determine the effects of SCL and CAI on anatomy performance outcomes. A moderator analysis of study features was also performed. Of the 3,035 records screened, 327 underwent full-text review. Seven studies, which comprised 1,564 participants, were included in the SCL analysis. An additional 19 studies analyzed the effects of CAI in the context of 2,570 participants. Upon comparing SCL to traditional instruction, a small positive effect on learner performance was detected (standardized mean difference (SMD = 0.24; [CI = 0.07, 0.42]; P = 0.006). Likewise, students with CAI exposure moderately outscored those with limited or no access to CAI (SMD = 0.59; [CI = 0.20, 0.98]; P = 0.003). Further analysis of CAI studies identified effects (P ≤ 0.001) for learner population, publication period, interventional approach, and intervention frequency. Overall, learners exposed to SCL and supplemental CAI outperformed their more classically-trained peers as evidenced by increases in short-term knowledge gains. Anat Sci Educ. © 2018 American Association of Anatomists.

Discovery and Structure-Activity-Relationship Study of N-Alkyl-5-hydroxypyrimidinone Carboxamides as Novel Antitubercular Agents Targeting Decaprenylphosphoryl-ß-d-ribose 2'-Oxidase.

Magnesium plays an important role in infection with Mycobacterium tuberculosis ( Mtb) as a signal of the extracellular environment, as a cofactor for many enzymes, and as a structural element in important macromolecules. Raltegravir, an antiretroviral drug that inhibits HIV-1 integrase is known to derive its potency from selective sequestration of active-site magnesium ions in addition to binding to a hydrophobic pocket. In order to determine if essential Mtb-related phosphoryl transfers could be disrupted in a similar manner, a directed screen of known molecules with integrase inhibitor-like pharmacophores ( N-alkyl-5-hydroxypyrimidinone carboxamides) was performed. Initial hits afforded compounds with low-micromolar potency against Mtb, acceptable cytotoxicity and PK characteristics, and robust SAR. Elucidation of the target of these compounds revealed that they lacked magnesium dependence and instead disappointingly inhibited a known promiscuous target in Mtb, decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1, Rv3790).

A meta-analysis of anatomy laboratory pedagogies.

The debate regarding anatomy laboratory teaching approaches is ongoing and controversial. To date, the literature has yielded only speculative conclusions because of general methodological weaknesses and a lack of summative empirical evidence. Through a meta-analysis, this study compared the effectiveness of instructional laboratory approaches used in anatomy education to objectively and more conclusively synthesize the existing literature. Studies published between January 1965 and December 2015 were searched through five databases. Titles and abstracts of the retrieved records were screened using eligibility criteria to determine their appropriateness for study inclusion. Only numerical data were extracted for analysis. A summary effect size was estimated to determine the effects of laboratory pedagogies on learner performance and perceptions data were compiled to provide additional context. Of the 3,035 records screened, 327 underwent full-text review. Twenty-seven studies, comprising a total of 7,731 participants, were included in the analysis. The meta-analysis detected no effect (standardized mean difference = -0.03; 95% CI = -0.16 to 0.10; P = 0.62) on learner performance. Additionally, a moderator analysis detected no effects (P ≥ 0.16) for study design, learner population, intervention length, or specimen type. Across studies, student performance on knowledge examinations was equivalent regardless of being exposed to either dissection or another laboratory instructional strategy. This was true of every comparison investigated (i.e., dissection vs. prosection, dissection vs. digital media, dissection vs. models/modeling, and dissection vs. hybrid). In the context of short-term knowledge gains alone, dissection is no better, and no worse, than alternative instructional modalities. Clin. Anat. 31:122-133, 2018. © 2017 Wiley Periodicals, Inc.

Measurement and limitations of optical orbital angular momentum through corrected atmospheric turbulence.

In recent years, there have been a series of proposals to exploit the orbital angular momentum (OAM) of light for astronomical applications. The OAM of light potentially represents a new way in which to probe the universe. The study of this property of light entails the development of new instrumentation and problems which must be addressed. One of the key issues is whether we can overcome the loss of the information carried by OAM due to atmospheric turbulence. We experimentally analyze the effect of atmospheric turbulence on the OAM content of a signal over a range of realistic turbulence strengths typical for astronomical observations. With an adaptive optics system we are able to recover up to 89% power in an initial non-zero OAM mode (ℓ = 1) at low turbulence strengths (0.30" FWHM seeing). However, for poorer seeing conditions (1.1" FWHM seeing), the amount of power recovered is significantly lower (5%), showing that for the terrestrial detection of astronomical OAM, a careful design of the adaptive optics system is needed.

Metronidazole prevents reactivation of latent Mycobacterium tuberculosis infection in macaques.

Targeting Mycobacterium tuberculosis bacilli in low-oxygen microenvironments, such as caseous granulomas, has been hypothesized to have the potential to shorten therapy for active tuberculosis (TB) and prevent reactivation of latent infection. We previously reported that upon low-dose M. tuberculosis infection, equal proportions of cynomolgus macaques develop active disease or latent infection and that latently infected animals reactivated upon neutralization of TNF. Using this model we now show that chemoprophylaxis of latently infected cynomolgus macaques with 6 mo of isoniazid (INH) effectively prevented anti-TNF antibody-induced reactivation. Similarly, 2-mo treatment of latent animals with a combination of INH and rifampicin (RIF) was highly effective at preventing reactivation disease in this model. Metronidazole (MTZ), which has activity only against anaerobic, nonreplicating bacteria, was as effective as either of these treatments in preventing reactivation of latent infection. Because hypoxic lesions also occur during active TB, we further showed that addition of MTZ to INH/RIF effectively treated animals with active TB within 2 mo. Healing lesions were associated with distinct changes in cellular pathology, with a shift toward increasingly fibrotic and calcified lesions. Our data in the nonhuman primate model of active and latent TB supports targeting bacteria in hypoxic environments for preventing reactivation of latent infection and possibly shortening the duration of therapy in active TB.

Maximizing fluence rate and field uniformity of light blanket for intraoperative PDT.

A light blanket is designed with a system of cylindrically diffusing optical fibers, which are spirally oriented. This 25×30 cm rectangular light blanket is capable of providing uniform illumination during intraoperative photodynamic therapy. The flexibility of the blanket proves to be extremely beneficial when conforming to the anatomical structures of the patient being treated. Previous tests of light distribution from the blanket have shown significant loss of intensity with the length of the fiber. This can be improved through the use of an optical adaptor which will be able to match the numerical aperture of the laser source to the numerical aperture of the blanket fiber; thus transmitting a higher percentage of light.

Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis.

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD(+). This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO(2) incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope ((13)C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from (13)C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.