Examining the Psychological Measures and Feasibility of Recreational Trail Rides in Reducing Cravings for Addictive Substances among Veterans: A Replication Pilot Study.

INTRODUCTION: Equine-assisted services have been gaining popularity as complementary therapies for a variety of populations with a diversity of diagnoses. Interventions using equines might benefit the military Veteran population through building therapeutic alliance as well as reducing symptoms. This pilot observational study is primarily aimed to investigate safety, feasibility, and acceptability of equine-assisted services for Veterans with substance abuse disorders. It is the second pilot observational study, the first being completed in 2022. Secondary outcomes included recording the impact of trail riding on psychological measures among Veterans at a large Veterans Administration medical center. METHODS: The study included a total of 32 Veterans who participated in trail riding sessions from May 4, 2022 to August 31, 2022. The study utilized the Positive and Negative Affect Scale, Adult Hope Scale, and Craving Experience Questionnaire to assess changes in positive affect, negative affect, hope components, and substance craving. RESULTS: The results indicated significant reductions in negative affect and substance craving after the first session, with positive effects observed in subsequent sessions. However, demographic and diagnostic variables did not predict significant changes in psychological instruments. CONCLUSION: Overall, the study demonstrated that trail riding was a safe and feasible intervention and may have potential benefits for improving emotional well-being and reducing substance cravings among Veterans. It is recommended that future studies research mechanisms of action, as well as conduct more rigorous investigations, including controlled trials using larger sample sizes and longitudinal research, to best determine lasting effects of these types of complementary therapies.

A Pilot Replication Study of Implementing an Equine-Assisted Services Program Within a VA Residential Substance Use Disorder Treatment Program.

INTRODUCTION: Veterans have high rates of substance use disorders and other mental health conditions including post-traumatic stress disorder. Effective treatments for these conditions exist; however, high attrition rates and residual symptoms after completing treatment are common. Complementary treatment approaches could enhance treatment engagement and/or response among this population. We previously reported a study of one such intervention, an equine-assisted learning, and psychotherapy incorporating horses intervention provided to veterans admitted to a Veterans Health Care Administration residential substance abuse treatment program. The first aim of this study was to replicate the previous study assessing the safety, feasibility, and preliminary outcomes of this intervention. The second aim was to examine the effect of participants attending multiple intervention sessions. MATERIALS AND METHODS: Participants were 94 veterans who participated in one to six sessions of a 3-4-hour program consisting of both equine-assisted learning and psychotherapy incorporating horses. Pre- and post-session administration of the Positive and Negative Affect Scale, State-Trait Anxiety Inventory, and Craving Experience Questionnaire was utilized to assess changes in affect, anxiety, and craving. Wilcoxon signed-rank or paired two-tailed t-tests were utilized for pre- to post-session comparisons of the outcome measures for sessions 1-4. Generalized linear mixed-effects (GLME) models were constructed to determine the impact of dosage. GLME models were constructed to determine the impact of dosage. RESULTS: As with our previous study, the intervention was safe and feasible to utilize for this population. There were statistically significant pre- to post-session improvements, with medium-to-large effect sizes, for sessions 1-3 for negative affect and sessions 1 and 2 for positive affect, anxiety, and craving. The GLME models revealed no statistical significance for any of the predictors. CONCLUSIONS: Taken together, this study and our previous investigation of this equine-assisted services intervention suggest that it is safe and feasible to utilize for veterans admitted to a residential substance abuse treatment program and we have now found short-term benefits in two separate studies. Thus, a randomized controlled trial of this intervention is warranted to demonstrate cause and effect and determine whether longer-term benefits are associated with the intervention. The finding that there was no additional benefit from attendance at more than two intervention sessions suggests that dose-response relationship studies of equine-assisted services interventions for veterans are needed.

Field Evaluation of an Oviposition Deterrent for Management of Spotted-Wing Drosophila, Drosophila suzukii, and Potential Nontarget Effects.

Spotted-wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), is a polyphagous, invasive pest of small fruits. Current management relies heavily on chemical insecticides, and an effective oviposition deterrent could contribute to alternative management approaches that reduce the need for these chemical insecticides. A novel deployment method for repelling Drosophila suzukii, thereby reducing D. suzukii oviposition in fall-bearing red raspberry, was evaluated in the field. Infestations occurring within 4 d after deployment were significantly lower in 2-m-long plots (Rubus idaeus 'Caroline') treated with the repellent (20% 1-octen-3-ol in specialized pheromone and lure application technology [SPLAT]) compared to control plots (blank SPLAT). Repellent-treated plots had roughly 28.8 and 49.5% fewer offspring reared per gram of fruit than control plots in two experiments, respectively. Nontarget effects were also evaluated in 2-m plot experiments as well as 5- by 5-m plot experiments. There were no differences in the number of parasitic hymenoptera trapped on yellow sticky cards hung in repellent compared to control plots. While there were no differences in the number of visits to raspberry flowers observed by honey bees in repellent versus control plots, the number of visits by bumble bees was greater in repellent plots compared to control plots. Challenges regarding evaporation rates and potential uses for repellents in an integrated pest management program for the control of D. suzukii are discussed.

Landscape Simplification Constrains Adult Size in a Native Ground-Nesting Bee.

Bees provide critical pollination services to 87% of angiosperm plants; however, the reliability of these services may become threatened as bee populations decline. Agricultural intensification, resulting in the simplification of environments at the landscape scale, greatly changes the quality and quantity of resources available for female bees to provision their offspring. These changes may alter or constrain the tradeoffs in maternal investment allocation between offspring size, number and sex required to maximize fitness. Here we investigate the relationship between landscape scale agricultural intensification and the size and number of individuals within a wild ground nesting bee species, Andrena nasonii. We show that agricultural intensification at the landscape scale was associated with a reduction in the average size of field collected A. nasonii adults in highly agricultural landscapes but not with the number of individuals collected. Small females carried significantly smaller (40%) pollen loads than large females, which is likely to have consequences for subsequent offspring production and fitness. Thus, landscape simplification is likely to constrain allocation of resources to offspring through a reduction in the overall quantity, quality and distribution of resources.

Ion channel mechanisms related to sudden cardiac death in phenotype-negative long-QT syndrome genotype-phenotype correlations of the KCNQ1(S349W) mutation.

UNLABELLED: BACKGROUND: Data regarding possible ion channel mechanisms that predispose to ventricular tachyarrhythmias in patients with phenotype-negative long-QT syndrome (LQTS) are limited. METHODS AND RESULTS: We carried out cellular expression studies for the S349W mutation in the KCNQ1 channel, which was identified in 15 patients from the International LQTS Registry who experienced a high rate of cardiac events despite lack of significant QTc prolongation. The clinical outcome of S349W mutation carriers was compared with that of QTc-matched carriers of haploinsufficient missense (n = 30) and nonsense (n = 45) KCNQ1 mutations. The channels containing the mutant S349W subunit showed a mild reduction in current (<50%), in the haploinsuficient range, with an increase in maximal conductance compared with wild-type channels. In contrast, expression of the S349W mutant subunit produced a pronounced effect on both the voltage dependence of activation and the time constant of activation, while haploinsuficient channels showed no effect on either parameter. The cumulative probability of cardiac events from birth through age 20 years was significantly higher among S349W mutation carriers (58%) as compared with carriers of QTc-matched haploinsufficent missense (21%, P = 0.004) and nonsense (25%, P = 0.01) mutations. CONCLUSIONS: The S349W mutation in the KCNQ1 potassium channel exerts a relatively mild effect on the ion channel current, whereas an increase in conductance compensates for impaired voltage activation of the channel. The changes observed in voltage activation of the channel may underlie the mechanisms predisposing to arrhythmic risk among LQTS patients with a normal-range QTc.

Molecular basis of decreased Kir4.1 function in SeSAME/EAST syndrome.

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Determination of Fab-hinge disulfide connectivity in structural isoforms of a recombinant human immunoglobulin G2 antibody.

The detection and characterization of unexpected disulfide-mediated structural variants of human immunoglobulin G2 (IgG2) antibodies was recently the subject of two copublications. In this paper, we present data to confirm the previously reported structures and elucidate the complete disulfide connectivity of each variant through the application of a novel analytical methodology. In this manner, the data illustrate the presence of at least five structural variants, including the classical structure with independent Fab domains and a hinge region. Multiple subvariants of the IgG2-A/B and IgG2-B structures are identified; these subvariants of each structure differ through the order of attachment of Fab peptides to the sequential hinge cysteines. Furthermore, the connectivity of a novel subvariant of IgG2-B containing an intrachain disulfide linkage in the lower hinge region is elucidated. The results presented in this paper reveal that the population of IgG2 disulfide structural variants is yet more complex than recently reported.

PTMSearchPlus: software tool for automated protein identification and post-translational modification characterization by integrating accurate intact protein mass and bottom-up mass spectrometric data searches.

PTMSearchPlus is a software tool for the automated integration of accurate intact protein mass (AIPM) and bottom-up (BU) mass spectra searches/data in order to both confidently identify the intact proteins and to characterize their post-translational modifications (PTMs). The development of PTMSearchPlus was motivated by the desire to effectively integrate high-resolution intact protein molecular masses with bottom-up peptide MS/MS data. PTMSearchPlus requires as input both intact protein and proteolytic peptide mass spectra collected from the same protein mixture, a FASTA protein database, and a selection of possible PTMs, the types and ranges of which can be specified. The output of PTMSearchPlus is a list of intact and modified proteins matching the AIPM data concomitant with their respective peptides found by the BU search. This list also contains protein and peptide sequence coverage information, scores, etc. that can be used for further evaluation or refiltering of the results. Corresponding and annotated AIPM and BU mass spectra are also displayed for visual inspection when a listed protein or a peptide is selected. These and other controls ensure that the user can manually evaluate, modify (e.g., remove obvious false positives, low quality spectra etc.), and save the results of the automated search if necessary. Driven by the exponential growth in the number of possible peptide candidates in a BU search when multiple PTMs are probed, the advantages on search speed by limiting the total number of possible PTMs on a peptide in the BU search or by performing an "AIPM predicted" BU search are also discussed in addition to the integration approach. The features of PTMSearchPlus are demonstrated using both a protein standard mixture and a complex protein mixture from Escherichia coli. Experimental data revealed a unique advantage of coupling AIPM and the BU data sets that is mutually beneficial for both approaches. Namely, AIPM data can confirm that no PTM peptides were missed in a BU search, while the BU search determines the location of the PTM. This information is not available using an AIPM search alone.

High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry.

We present an optimized high-throughput method for the characterization of 2-aminobenzamide (2-AB)-labeled N-glycans from recombinant immunoglobulin G (rIgG). This method includes an optimized sample preparation protocol involving microwave-assisted deglycosylation in conjunction with an automated sample cleanup strategy and a rapid resolution reverse-phase high-performance liquid chromatography (RRRP-HPLC) separation of labeled N-glycans. The RRRP-HPLC method permits generation of a comprehensive glycan profile using fluorescence detection in 45min. In addition, the profiling method is directly compatible with electrospray ionization mass spectrometry (ESI-MS), allowing immediate and sensitive characterization of the glycan moiety by intact MS and tandem MS (MS/MS) fragmentation. We conservatively estimate an efficiency gain of fourfold with respect to the throughput capabilities of this optimized method as compared with traditional protocols (overnight deglycosylation, sample cleanup by graphitized carbon or cellulose cartridge, high-pH anion exchange chromatography, fraction collection, and processing for matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] MS analysis) for a single sample. Even greater gains are achieved when processing of multiple samples is considered.

Relationship of IQ to suicide and homicide rate: an international perspective.

IQs were correlated with the z score of suicide rate minus z score of homicide rate using nine regions of the world--established market economies, formerly socialized Europe, India, China, other Asian nations, Sub-Saharan Africa, Latin America/Caribbean, Middle East Crescent, and the USA. Correlations were .85 and .83 with crude rates and age-adjusted rates, respectively. The homicide findings are consistent with previous research in individual countries showing that less intelligent persons commit homicide more often. However, the present findings of a positive correlation between IQ and suicide rates are the opposite of what has been found in the more definitive studies within countries. Explanations for the apparent paradox and for the findings more generally were offered.

Characterization of pII family (GlnK1, GlnK2, and GlnB) protein uridylylation in response to nitrogen availability for Rhodopseudomonas palustris.

The GlnK and GlnB proteins are members of the pII signal transduction protein family, which is essential in nitrogen regulation due to this protein family's ability to sense internal cellular ammonium levels and control cellular response. The role of GlnK in nitrogen regulation has been studied in a variety of bacteria but previously has been uncharacterized in the purple nonsulfur anoxygenic phototropic bacterium Rhodopseudomonas palustris. R. palustris has tremendous metabolic versatility in its modes of energy generation and carbon metabolism, and it employs a sensitive nitrogen-ammonium regulation system that may vary from that of other commonly studied bacteria. In R. palustris, there are three annotated forms of pII proteins: GlnK1, GlnK2, and GlnB. Here we describe, for the first time, the characterization of GlnK1, GlnK2, and GlnB modifications as a response to nitrogen availability, thereby providing information about how this bacterium regulates the AmtB ammonium transporter and glutamine synthetase, which controls the rate of glutamate to glutamine conversion. Using a strategy of creating C-terminally tagged GlnK and GlnB proteins followed by tandem affinity purification in combination with top-down mass spectrometry, four isoforms of the GlnK2 and GlnB proteins and two isoforms of the GlnK1 protein were characterized at high resolution and mass accuracy. Wild-type or endogenous expression of all three proteins was also examined under normal ammonium conditions and ammonium starvation to ensure that the tagging and affinity purification methods employed did not alter the natural state of the proteins. All three proteins were found to undergo uridylylation under ammonium starvation conditions, presumably to regulate the AmtB ammonium transporter and glutamine synthetase. Under high-ammonium conditions, the GlnK1, GlnK2, and GlnB proteins are unmodified. This experimental protocol involving high-resolution mass spectrometry measurements of intact proteins provides a powerful method of examining the posttranslational modifications that play a crucial role in both the regulation of the AmtB ammonium transporter and glutamine synthetase within R. palustris.

Determination of peptide and protein ion charge states by Fourier transformation of isotope-resolved mass spectra.

We report an automated method for determining charge states from high-resolution mass spectra. Fourier transforms of isotope packets from high-resolution mass spectra are compared to Fourier transforms of modeled isotopic peak packets for a range of charge states. The charge state for the experimental ion packet is determined by the model isotope packet that yields the best match in the comparison of the Fourier transforms. This strategy is demonstrated for determining peptide ion charge states from "zoom scan" data from a linear quadrupole ion trap mass spectrometer, enabling the subsequent automated identification of singly- through quadruply-charged peptide ions, while reducing the numbers of conflicting identifications from ambiguous charge state assignments. We also apply this technique to determine the charges of intact protein ions from LC-FTICR data, demonstrating that it is more sensitive under these experimental conditions than two existing algorithms. The strategy outlined in this paper should be generally applicable to mass spectra obtained from any instrument capable of isotopic resolution.

Transcriptomic and proteomic characterization of the Fur modulon in the metal-reducing bacterium Shewanella oneidensis.

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach.

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.

Mass spectrometric approaches for characterizing bacterial proteomes.

The emergence of advanced liquid chromatography mass spectrometry technologies for characterizing very complex mixtures of proteins has greatly propelled the field of proteomics, the goal of which is the simultaneous examination of all the proteins expressed by an organism. This research area represents a paradigm shift in molecular biology by attempting to provide a top-down qualitative and quantitative view of all the proteins (including their modifications and interactions) that are essential for an organism's life cycle, rather than targeting a particular protein family. This level of global protein information about an organism such as a bacterium can be combined with genomic and metabolomic data to enable a systems biology approach for understanding how these organisms live and function.