Pre-analytical challenges from adsorptive losses associated with thiamine analysis.

Thiamine (vitamin B1) is an essential vitamin serving in its diphosphate form as a cofactor for enzymes in the citric acid cycle and pentose-phosphate pathways. Its concentration reported in the pM and nM range in environmental and clinical analyses prompted our consideration of the components used in pre-analytical processing, including the selection of filters, filter apparatuses, and sample vials. The seemingly innocuous use of glass fiber filters, glass filter flasks, and glass vials, ubiquitous in laboratory analysis of clinical and environmental samples, led to marked thiamine losses. 19.3 nM thiamine was recovered from a 100 nM standard following storage in glass autosampler vials and only 1 nM of thiamine was obtained in the filtrate of a 100 nM thiamine stock passed through a borosilicate glass fiber filter. We further observed a significant shift towards phosphorylated derivatives of thiamine when an equimolar mixture of thiamine, thiamine monophosphate, and thiamine diphosphate was stored in glass (most notably non-silanized glass, where a reduction of 54% of the thiamine peak area was observed) versus polypropylene autosampler vials. The selective losses of thiamine could lead to errors in interpreting the distribution of phosphorylated species in samples. Further, some loss of phosphorylated thiamine derivatives selectively to amber glass vials was observed relative to other glass vials. Our results suggest the use of polymeric filters (including nylon and cellulose acetate) and storage container materials (including polycarbonate and polypropylene) for thiamine handling. Losses to cellulose nitrate and polyethersulfone filters were far less substantial than to glass fiber filters, but were still notable given the low concentrations expected in samples. Thiamine losses were negated when thiamine was stored diluted in trichloroacetic acid or as thiochrome formed in situ, both of which are common practices, but not ubiquitous, in thiamine sample preparation.

Understanding and mitigating thiaminase activity in silver carp.

A deficiency of thiamine (vitamin B1), an essential cofactor for enzymes involved in metabolic processes, can be caused by the enzyme thiaminase. Thiaminase in food stocks has been linked to morbidity and mortality due to thiamine depletion in many ecologically and economically important species. Thiaminase activity has been detected in certain bacteria, plants, and fish species, including carp. The invasive silver carp (Hypophthalmichthys molitrix) presents an enormous burden to ecosystems throughout the Mississippi River watershed. Its large biomass and nutritional content offer an attractive possibility as a food source for humans, wild animals, or pets. Additionally, harvesting this fish could alleviate some of the effects of this species on waterways. However, the presence of thiaminase would detract from its value for dietary consumption. Here we confirm the presence of thiaminase in several tissues from silver carp, most notably the viscera, and systematically examine the effects of microwaving, baking, dehydrating, and freeze-drying on thiaminase activity. Certain temperatures and durations of baking and microwaving reduced thiaminase activity to undetectable levels. However, caution should be taken when carp tissue is concentrated by processes without sufficient heat treatment, such as freeze-drying or dehydration, which results in concentration, but not inactivation of the enzyme. The effects of such treatments on the ease of extracting proteins, including thiaminase, and the impact on data interpretation using the 4-nitrothiophenol (4-NTP) thiaminase assay were considered.

Dietary factors potentially impacting thiaminase I-mediated thiamine deficiency.

Fish population declines from thiamine (vitamin B1) deficiency have been widespread in ecologically and economically valuable organisms, ranging from the Great Lakes to the Baltic Sea and, most recently, the California coast. Thiamine deficiencies in predatory fishes are often attributed to a diet of prey fishes with high levels of thiamine-degrading (e.g., thiaminase) enzymes, such as alewives, rainbow smelt, and anchovies. Since their discovery, thiaminase I enzymes have been recognized for breaking down thiamine into its pyrimidine and thiazole moieties using various nucleophilic co-substrates to afford cleavage, but these studies have not thoroughly considered other factors that could modify enzyme activity. We found the thiaminase I enzyme from Clostridium botulinum efficiently degrades thiamine in the presence of pyridoxine (vitamin B6) as a co-substrate but has relatively limited activity in the presence of nicotinic acid (vitamin B3). Using fluorescence measurements, thiamine degradation in an over-the-counter complete multivitamin formulation was inhibited, and a B-complex formulation required co-substrate supplementation for maximal thiamine depletion. These studies prompted the evaluation of specific constituents contributing to thiaminase I inhibition by both chromatography and fluorescence assays: Cu2+ potently and irreversibly inhibited thiamine degradation; ascorbic acid was a strong but reversible inhibitor; Fe2+, Mn2+ and Fe3+ modulated thiamine degradation to a lesser degree. The enhancement by pyridoxine and inhibition by Cu2+ extended to thiaminase-mediated degradation from Burkholderia thailandensis, Paenibacillus thiaminolyticus, and Paenibacillus apiarius in tryptic soy broth supernatants. These co-substrate limitations and the common presence of inhibitory dietary factors complement recent studies reporting that the intended function of thiaminase enzymes is to recycle thiamine breakdown products for thiamine synthesis, not thiamine degradation.

Serum GFAP, NfL, and tau concentrations are associated with worse neurobehavioral functioning following mild, moderate, and severe TBI: a cross-sectional multiple-cohort study.

Introduction: The purpose of this study was to examine whether blood-based biomarkers associate with neurobehavioral functioning at three time points following traumatic brain injury (TBI). Materials and methods: Participants were 328 United States service members and veterans (SMVs) prospectively enrolled in the Defense and Veterans Brain Injury Center-Traumatic Brain Injury Center of Excellence (DVBIC-TBICoE) 15-Year Longitudinal TBI Study, recruited into three groups: uncomplicated mild TBI (MTBI, n = 155); complicated mild, moderate, severe TBI combined (STBI, n = 97); non-injured controls (NIC, n = 76). Participants were further divided into three cohorts based on time since injury (≤12 months, 3-5 years, and 8-10 years). Participants completed the Minnesota Multiphasic Personality Inventory-2-Restructured Format (MMPI-2-RF) and underwent blood draw to measure serum concentrations of glial fibrillary acidic protein (GFAP), neurofilament light (NfL), and tau. A total of 11 MMPI-2-RF scales were examined (e.g., depression, anxiety, anger, somatic, cognitive symptoms). Stepwise hierarchical regression models were conducted within each group. Results: Significant associations were found between biomarkers and MMPI-2-RF scales (all p < 0.05; R2Δ > 0.10). GFAP was inversely related to (a) neurological complaints in the MTBI group at ≤12 months, (b) demoralization, anger proneness in the STBI group at ≤12 months, and (c) head pain complaints in the STBI group at 8-10 years. NfL was (a) related to low positive emotions in the NIC group; and inversely related to (b) demoralization, somatic complaints, neurological complaints, cognitive complaints in the MTBI group at ≤12 months, (c) demoralization in the STBI group at ≤12 months, and (d) demoralization, head pain complaints, stress/worry in the STBI group at 3-5 years. In the STBI group, there were meaningful findings (R2Δ > 0.10) for tau, NFL, and GFAP that did not reach statistical significance. Discussion: Results indicate worse scores on some MMPI-2-RF scales (e.g., depression, stress/worry, neurological and head pain complaints) were associated with lower concentrations of serum GFAP, NfL, and tau in the sub-acute and chronic phase of the recovery trajectory up to 5 years post-injury, with a reverse trend observed at 8-10 years. Longitudinal studies are needed to help elucidate any patterns of association between blood-based biomarkers and neurobehavioral outcome over the recovery trajectory following TBI.

Cytokine Profiles Differentiate Symptomatic from Asymptomatic PTSD in Service Members and Veterans with Chronic Traumatic Brain Injury.

Traumatic brain injuries (TBI) and posttraumatic stress disorder (PTSD) are commonly observed comorbid occurrences among military service members and veterans (SMVs). In this cross-sectional study, SMVs with a history of TBI were stratified into symptomatic and asymptomatic PTSD groups based on posttraumatic stress checklist-civilian (PCL-C) total scores. Blood-based biomarkers were assessed, and significant differential markers were associated with scores from multiple neurobehavioral self-report assessments. PCL-C cutoffs were total scores >50 (PTSD symptomatic) and <25 (asymptomatic). Cytokines IL6, IL8, TNFα, and IL10 were significantly elevated (p < 0.05−0.001) in the TBI+/PTSD symptomatic group compared to the TBI+/asymptomatic group. Cytokine levels of IL8, TNFα, and IL10 were strongly associated with PCL-C scores (0.356 < r > 0.624 for all, p < 0.01 for all), while TNFα and IL10 were additionally associated with NSI totals (r = 0.285 and r = 0.270, p < 0.05, respectively). This is the first study focused on PTSD symptom severity to report levels of circulating pro-inflammatory IL8, specifically in SMVs with TBI. These data suggest that within the military TBI population, there are unique cytokine profiles that relate to neurobehavioral outcomes associated with TBI and PTSD.

Elevations in Tumor Necrosis Factor Alpha and Interleukin 6 From Neuronal-Derived Extracellular Vesicles in Repeated Low-Level Blast Exposed Personnel.

Objective: The purpose of this pilot study was to determine if military service members with histories of hundreds to thousands of low-level blast exposures (i. e., experienced breachers) had different levels of serum and neuronal-derived extracellular vesicle (EV) concentrations of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNFα), compared to matched controls, and if these biomarkers related to neurobehavioral symptoms. Methods: Participants were experienced breachers (n = 20) and matched controls without blast exposures (n = 14). Neuronal-derived EVs were isolated from serum and identified with mouse anti-human CD171. Serum and neuronal-derived EVs were analyzed for IL-6, IL-10, and TNFα using an ultra-sensitive assay. Results: Serum TNFα concentrations were decreased in breachers when compared to control concentrations (p < 0.01). There were no differences in serum concentrations of IL-6, IL-10, or the IL-6/IL-10 ratio between breachers and controls (p's > 0.01). In neuronal-derived EVs, TNFα and IL-6 levels were increased in breachers compared to controls (p's < 0.01), and IL-10 levels were decreased in the breacher group compared to controls (p < 0.01). In breachers the IL-6/IL-10 ratio in neuronal-derived EVs was higher compared to controls, which correlated with higher total Rivermead Post-concussion Questionnaire (RPQ) scores (p's < 0.05). Conclusions: These findings suggest that exposure of personnel to high numbers of low-level blast over a career may result in enduring central inflammation that is associated with chronic neurological symptoms. The data also suggest that peripheral markers of inflammation are not necessarily adequate surrogates for central neuroinflammation.

A Pilot Study of Whole-Blood Transcriptomic Analysis to Identify Genes Associated with Repetitive Low-Level Blast Exposure in Career Breachers.

Repetitive low-level blast exposure is one of the major occupational health concerns among US military service members and law enforcement. This study seeks to identify gene expression using microRNA and RNA sequencing in whole-blood samples from experienced breachers and unexposed controls. We performed experimental RNA sequencing using Illumina's HiSeq 2500 Sequencing System, and microRNA analysis using NanoString Technology nCounter miRNA expression panel in whole-blood total RNA samples from 15 experienced breachers and 14 age-, sex-, and race-matched unexposed controls. We identified 10 significantly dysregulated genes between experienced breachers and unexposed controls, with FDR corrected <0.05: One upregulated gene, LINC00996 (long intergenic non-protein coding RNA 996); and nine downregulated genes, IGLV3-16 (immunoglobulin lambda variable 3-16), CD200 (CD200 molecule), LILRB5 (leukocyte immunoglobulin-like receptor B5), ZNF667-AS1 (ZNF667 antisense RNA 1), LMOD1 (leiomodin 1), CNTNAP2 (contactin-associated protein 2), EVPL (envoplakin), DPF3 (double PHD fingers 3), and IGHV4-34 (immunoglobulin heavy variable 4-34). The dysregulated gene expressions reported here have been associated with chronic inflammation and immune response, suggesting that these pathways may relate to the risk of lasting neurological symptoms following high exposures to blast over a career.

Sex Differences in Behavioral Symptoms and the Levels of Circulating GFAP, Tau, and NfL in Patients With Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of Americans each year and has been shown to disproportionately impact those subject to greater disparities in health. Female sex is one factor that has been associated with disparities in health outcomes, including in TBI, but sex differences in biomarker levels and behavioral outcomes after TBI are underexplored. This study included participants with both blunt and blast TBI with majority rating their TBI as mild. Time since injury was 5.4 (2.0, 15.5) years for females and 6.8 (2.4, 11.3) years for males. The aim of this cross sectional study is to investigate the relationship between postconcussive, depression, and post-traumatic stress disorder (PTSD) symptoms, as well as health related quality of life (HRQOL), and the levels of glial fibrillary acidic protein (GFAP), total tau (t-tau), neurofilament light chain (NfL), and ubiquitin C-terminal hydrolase-L1 (UCH-L1). Behavioral outcomes were evaluated with the Neurobehavioral Symptom Inventory (NSI), Patient Health Questionnaire-9 (PHQ-9), PTSD Checklist- Civilian Version (PCL-C), short form (SF)-36, and plasma levels of total tau, GFAP, NfL, and UCHL-1 measured with the Simoa-HDX. We observed that females had significantly higher levels of GFAP and tau (ps < 0.05), and higher PHQ-9 scores, NSI total scores, NSI- vestibular, NSI-somatosensory, NSI-affective sub-scale scores (ps < 0.05)), than males. In addition, females had lower scores in HRQOL outcomes of role limitations due to emotional problems, vitality, emotional well-being, social functioning, and pain compared to males (ps < 0.05). Correlation analysis showed positive associations between levels of tau and the NSI-total and NSI-cognitive sub-scale scores (ps < 0.05) in females. No significant associations were found for NfL or GFAP with NSI scores. For female participants, negative correlations were observed between tau and NfL concentrations and the SF-36 physical function subscale (ps < 0.05), as well as tau and the social function subscale (p < 0.001), while GFAP levels positively correlated with role limitations due to emotional problems (p = 0.004). No significant associations were observed in males. Our findings suggest that sex differences exist in TBI-related behavioral outcomes, as well as levels of biomarkers associated with brain injury, and that the relationship between biomarker levels and behavioral outcomes is more evident in females than males. Future studies are warranted to corroborate these results, and to determine the implications for prognosis and treatment. The identification of candidate TBI biomarkers may lead to development of individualized treatment guidelines.

Extracellular Vesicle Proteins and MicroRNAs Are Linked to Chronic Post-Traumatic Stress Disorder Symptoms in Service Members and Veterans With Mild Traumatic Brain Injury.

Symptoms of post-traumatic stress disorder (PTSD) are common in military populations, and frequently associated with a history of combat-related mild traumatic brain injury (mTBI). In this study, we examined relationships between severity of PTSD symptoms and levels of extracellular vesicle (EV) proteins and miRNAs measured in the peripheral blood in a cohort of military service members and Veterans (SMs/Vs) with chronic mTBI(s). Participants (n = 144) were divided into groups according to mTBI history and severity of PTSD symptoms on the PTSD Checklist for DSM-5 (PCL-5). We analyzed EV levels of 798 miRNAs (miRNAs) as well as EV and plasma levels of neurofilament light chain (NfL), Tau, Amyloid beta (Aß) 42, Aß40, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNFα), and vascular endothelial growth factor (VEGF). We observed that EV levels of neurofilament light chain (NfL) were elevated in participants with more severe PTSD symptoms (PCL-5 ≥ 38) and positive mTBI history, when compared to TBI negative controls (p = 0.024) and mTBI participants with less severe PTSD symptoms (p = 0.006). Levels of EV NfL, plasma NfL, and hsa-miR-139-5p were linked to PCL-5 scores in regression models. Our results suggest that levels of NfL, a marker of axonal damage, are associated with PTSD symptom severity in participants with remote mTBI. Specific miRNAs previously linked to neurodegenerative and inflammatory processes, and glucocorticoid receptor signaling pathways, among others, were also associated with the severity of PTSD symptoms. Our findings provide insights into possible signaling pathways linked to the development of persistent PTSD symptoms after TBI and biological mechanisms underlying susceptibility to PTSD.

Neuronally-derived tau is increased in experienced breachers and is associated with neurobehavioral symptoms.

Military and law enforcement breachers are exposed to many low-level blasts during their training and occupational experiences in which they detonate explosives to force entry into secured structures. There is a concern that exposure to these repetitive blast events in career breachers could result in cumulative neurological effects. This study aimed to determine concentrations of neurofilament light (NF-L), tau, and amyloid-beta 42 (Aß42) in serum and in neuronal-derived extracellular vesicles (EVs) in an experienced breacher population, and to examine biomarker associations with neurobehavioral symptoms. Thirty-four participants enrolled in the study: 20 experienced breachers and 14 matched military or civilian law enforcement controls. EV tau concentrations were significantly elevated in experienced breachers (0.3301 ± 0.5225) compared to controls (-0.4279 ± 0.7557; F = 10.43, p = 0.003). No statistically significant changes were observed in EV levels of NF-L or Aß42 or in serum levels of NF-L, tau, or Aß42 (p's > 0.05). Elevated EV tau concentrations correlated with increased Neurobehavioral Symptom Inventory (NSI) score in experienced breachers (r = 0.596, p = 0.015) and predicted higher NSI score (F(1,14) = 7.702, p = 0.015, R2 = 0.355). These findings show that neuronal-derived EV concentrations of tau are significantly elevated and associated with neurobehavioral symptoms in this sample of experienced breachers who have a history of many low-level blast exposures.

Technical considerations to development of serological tests for SARS-CoV-2.

The COVID-19 pandemic has had a devastating impact worldwide and has brought clinical assays both for acute diagnosis and prior exposure determination to the forefront. Serological testing intended for point-of-care or laboratory use can be used to determine more accurate individual and population assessments of prior exposure to SARS-CoV-2; improve our understanding of the degree to which immunity is conveyed to subsequent exposures; and quantify immune response to future vaccines. In response to this pandemic, initially more than 90 companies deployed serology assays to the U.S. market, many of which made overstated claims for their accuracy, regulatory approval status, and utility for intended purpose. The U.S. Food and Drug Administration subsequently instituted an Emergency Use Authorization (EUA) procedure requiring that manufacturers submit validation data, but allowing newly developed serological tests to be marketed without the usual approval process during this crisis. Although this rapid deployment was intended to benefit public health, the incomplete understanding of immune response to the virus and lack of assay vetting resulted in quality issues with some of these tests, and thus many were withdrawn after submission. Common assay platforms include lateral flow assays which can serve an important niche of low cost, rapid turnaround, and increased accessibility whereas established laboratory-based platforms based on ELISAs and chemiluminescence expand existing technologies to SARS-CoV-2 and can provide throughput and quantification capabilities. While most of the currently EUA assays rely on these well-established platforms, despite their apparent technical simplicity, there are numerous practical challenges both for manufacturers in developing and for end-users in running and interpreting such assays. Within are discussed technical challenges to serology development for SARS-CoV-2, with an emphasis on lateral flow assay technology.

Blast exposure results in tau and neurofilament light chain changes in peripheral blood.

OBJECTIVES: To evaluate how blast exposure impacts peripheral biomarkers.in military personnel enrolled in 10-day blast training. METHODS: On day 7, 21 military personnel experienced peak overpressure <2 pounds per square inch (psi); while 29 military personnel experienced peak overpressure ≥5 psi. Blood samples were collected each day to measure changes in amyloid beta (Aß), neurofilament light chain (NFL), and tau concentrations. RESULTS: Within 24 hours following exposure ≥5 psi, the ≥5 psi group had lower Aß42 (p = .004) and NFL (p < .001) compared to the <2 psi group and lower Aß42 (9.35%) and NFL (22.01%) compared to baseline. Twenty-four hours after ≥5 psi exposure, the ≥5 psi group had lower tau (p < .001) and NFL (p < .001) compared to the <2 psi group and baseline. Seventy-two hours after exposure ≥5 psi, tau increased in the ≥5 psi group compared to the <2 psi group (p = .02) and baseline. The tau:Aß42 ratio 24 hours after blast (p = .012), and the Aß40:Aß42 ratio 48 hours after blast (p = .04) differed in the ≥5 psi group compared to the <2 psi group. CONCLUSIONS: These findings provide an initial report of acute alterations in biomarker concentrations following blast exposure.

Inflammatory Cytokines Associate With Neuroimaging After Acute Mild Traumatic Brain Injury.

Introduction: Elevated levels of blood-based proinflammatory cytokines are linked to acute moderate to severe traumatic brain injuries (TBIs), yet less is known in acute mild (m)TBI cohorts. The current study examined whether blood-based cytokines can differentiate patients with mTBI, with and without neuroimaging findings (CT and MRI). Material and Methods: Within 24 h of a mTBI, determined by a Glasgow Coma Scale (GCS) between 13 and 15, participants (n = 250) underwent a computed tomography (CT) and magnetic resonance imaging (MRI) scan and provided a blood sample. Participants were classified into three groups according to imaging findings; (1) CT+, (2) MRI+ (CT-), (3) Controls (CT- MRI-). Plasma levels of circulating cytokines (IL-6, IL-10, TNFα), and vascular endothelial growth factor (VEGF) were measured using an ultra-sensitive immunoassay. Results: Concentrations of inflammatory cytokines (IL-6, TNFα) and VEGF were elevated in CT+, as well as MRI+ groups (p < 0.001), compared to controls, even after controlling for age, sex and cardiovascular disease (CVD)-related risk factors; hypertension, and hyperlipidemia. Post-concussive symptoms were associated with imaging groupings, but not inflammatory cytokines in this cohort. Levels of VEGF, IL-6, and TNFα differentiated patients with CT+ findings from controls, with the combined biomarker model (VEGF, IL-6, TNFα, and IL-10) showing good discriminatory power (AUC 0.92, 95% CI 0.87-0.97). IL-6 was a fair predictor of MRI+ findings compared to controls (AUC 0.70, 95% CI 0.60-0.78). Finally, the combined biomarker model discriminated patients with MRI+ from CT+ with an AUC of 0.71 (95% CI 0.62-0.80). Conclusions: When combined, IL-6, TNFα, and VEGF may provide a promising biomarker cytokine panel to differentiate mTBI patients with CT+ imaging vs. controls. Singularly, IL-6 was a fair discriminator between each of the imaging groups. Future research directions may help elucidate mechanisms related to injury severity and potentially, recovery following an mTBI.

Interleukin-6 is associated with acute concussion in military combat personnel.

BACKGROUND: Concussion is the most common type of TBI, yet reliable objective measures related to these injuries and associated recovery processes remain elusive, especially in military personnel. The purpose of this study was to characterize the relationship between cytokines and recovery from acute brain injury in active duty service members. Inflammatory cytokines (IL-6, IL-10, and TNFα) were measured acutely in blood samples within 8 h following a medically diagnosed concussion and then 24 h later. METHODS: Participants (n = 94) were categorized into two groups: 1) military personnel who sustained provider-diagnosed concussion, without other major medical diagnosis (n = 45) and 2) healthy control participants in the same deployment environment who did not sustain concussion or other illness or injuries (n = 49). IL-6, IL-10, and TNFα concentrations were measured using an ultrasensitive single-molecule enzyme-linked immunosorbent assay. Differences in cytokine levels between concussed and healthy groups were evaluated at two time points (time point 1 ≤ 8 h after injury; time point 2 = 24 h following time point 1). RESULTS: At time point 1, IL-6 median (IQR) concentrations were 2.62 (3.62) in the concussed group, which was greater compared to IL-6 in the healthy control group (1.03 (0.90); U = 420.00, z = - 5.12, p < 0.001). Compared to healthy controls, the concussed group did not differ at time point 1 in IL-10 or TNFα concentrations (p's > 0.05). At time point 2, no differences were detected between concussed and healthy controls for IL-6, IL-10, or TNFα (p's > 0.05). The median difference between time points 1 and 2 were compared between the concussed and healthy control groups for IL-6, IL-10, and TNFα. Change in IL-6 across time was greater for the concussed group than healthy control (- 1.54 (3.12); U = 315.00, z = - 5.96, p < 0.001), with no differences between groups in the change of IL-10 or TNFα (p's > 0.05). CONCLUSION: Reported here is a significant elevation of IL-6 levels in concussed military personnel less than 8 h following injury. Future studies may examine acute and chronic neurological symptomology associated with inflammatory cytokine levels, distinguish individuals at high risk for developing neurological complications, and identify underlying biological pathways to mitigate inflammation and improve outcomes.

The Photoconvertible Fluorescent Probe, CaMPARI, Labels Active Neurons in Freely-Moving Intact Adult Fruit Flies.

Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI's uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.

A Moderate Blast Exposure Results in Dysregulated Gene Network Activity Related to Cell Death, Survival, Structure, and Metabolism.

Blast exposure is common in military personnel during training and combat operations, yet biological mechanisms related to cell survival and function that coordinate recovery remain poorly understood. This study explored how moderate blast exposure influences gene expression; specifically, gene-network changes following moderate blast exposure. On day 1 (baseline) of a 10-day military training program, blood samples were drawn, and health and demographic information collected. Helmets equipped with bilateral sensors worn throughout training measured overpressure in pounds per square inch (psi). On day 7, some participants experienced moderate blast exposure (peak pressure ≥5 psi). On day 10, 3 days post-exposure, blood was collected and compared to baseline with RNA-sequencing to establish gene expression changes. Based on dysregulation data from RNA-sequencing, followed by top gene networks identified with Ingenuity Pathway Analysis, a subset of genes was validated (NanoString). Five gene networks were dysregulated; specifically, two highly significant networks: (1) Cell Death and Survival (score: 42), including 70 genes, with 50 downregulated and (2) Cell Structure, Function, and Metabolism (score: 41), including 69 genes, with 41 downregulated. Genes related to ubiquitination, including neuronal development and repair: UPF1, RNA Helicase and ATPase (UPF1) was upregulated while UPF3 Regulator of Nonsense Transcripts Homolog B (UPF3B) was downregulated. Genes related to inflammation were upregulated, including AKT serine/threonine kinase 1 (AKT1), a gene coordinating cellular recovery following TBIs. Moderate blast exposure induced significant gene expression changes including gene networks involved in (1) cell death and survival and (2) cellular development and function. The present findings may have implications for understanding blast exposure pathology and subsequent recovery efforts.

Assessing a Blast-Related Biomarker in an Operational Community: Glial Fibrillary Acidic Protein in Experienced Breachers.

Mild traumatic brain injury (mTBI) is a risk for military personnel due to blast overpressures, which may result from a variety of sources, including artillery and improvised explosive devices. Much research has gone into the search for a biomarker to identify patients with a TBI. The FDA recently identified two proteins, glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1), as biomarkers to evaluate suspected brain injury. Our group previously observed changes in UCH-L1 in a military population exposed to repeated blast. In our current study we assessed GFAP protein levels in a military population exposed to repeated blast during a 2-week training protocol. We observed GFAP levels were reduced in the moderate blast cases on days 6 and 7 during the training. Specifically, moderate blast cases showed a 24.07% reduction from baseline on day 6 and a 29.61% reduction on day 7. Further, GFAP levels were negatively correlated with cumulative blast experienced during training and with duration of military service. We observed that repeated blast exposure at low levels may impact acute changes in GFAP. Additionally subacute cumulative blast exposure or duration of service was also a factor in influencing GFAP levels.

Periplasmic binding protein-based magnetic isolation and detection of thiamine in complex biological matrices.

Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine periplasmic binding protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5 nM ranging up to 240 nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2 nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0 nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.

Thiaminase I Provides a Growth Advantage by Salvaging Precursors from Environmental Thiamine and Its Analogs in Burkholderia thailandensis.

Thiamine is essential to life, as it serves as a cofactor for enzymes involved in critical carbon transformations. Many bacteria can synthesize thiamine, while thiamine auxotrophs must obtain it or its precursors from the environment. Thiaminases degrade thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, and thiaminase I specifically has been implicated in thiamine deficiency syndromes in animals. The biological role of this secreted enzyme has been a long-standing mystery. We used the thiaminase I-producing soil bacterium Burkholderia thailandensis as a model to ascertain its function. First, we generated thiamine auxotrophs, which are still able to use exogenous precursors (thiazole and hydroxymethyl pyrimidine), to synthesize thiamine. We found that thiaminase I extended the survival of these strains, when grown in defined media where thiamine was serially diluted out, compared to isogenic strains that could not produce thiaminase I. Thiamine auxotrophs grew better on thiamine precursors than thiamine itself, suggesting thiaminase I functions to convert thiamine to useful precursors. Furthermore, our findings demonstrate that thiaminase I cleaves phosphorylated thiamine and toxic analogs, which releases precursors that can then be used for thiamine synthesis. This study establishes a biological role for this perplexing enzyme and provides additional insight into the complicated nature of thiamine metabolism and how individual bacteria may manipulate the availability of a vital nutrient in the environment.IMPORTANCE The function of thiaminase I has remained a long-standing, unsolved mystery. The enzyme is only known to be produced by a small subset of microorganisms, although thiaminase I activity has been associated with numerous plants and animals, and is implicated in thiamine deficiencies brought on by consumption of organisms containing this enzyme. Genomic and biochemical analyses have shed light on potential roles for the enzyme. Using the genetically amenable thiaminase I-producing soil bacterium Burkholderia thailandensis, we were able to demonstrate that thiaminase I helps salvage precursors from thiamine derivatives in the environment and degrades thiamine to its precursors, which are preferentially used by B. thailandensis auxotrophs. Our study establishes a biological role for this perplexing enzyme and provides insight into the complicated nature of thiamine metabolism. It also establishes B. thailandensis as a robust model system for studying thiamine metabolism.

A Kappa Opioid Receptor Agonist Blocks Bone Cancer Pain Without Altering Bone Loss, Tumor Size, or Cancer Cell Proliferation in a Mouse Model of Cancer-Induced Bone Pain.

Breast cancer metastasizes to bone, diminishing quality of life of patients because of pain, fracture, and limited mobility. Cancer-induced bone pain (CIBP) is characterized as moderate to severe ongoing pain, primarily managed by mu opioid agonists such as fentanyl. However, opioids are limited by escalating doses and serious side effects. One alternative may be kappa opioid receptor (KOR) agonists. There are few studies examining KOR efficacy on CIBP, whereas KOR agonists are efficacious in peripheral and inflammatory pain. We thus examined the effects of the KOR agonist U50,488 given twice daily across 7 days to block CIBP, tumor-induced bone loss, and tumor burden. U50,488 dose-dependently blocked tumor-induced spontaneous flinching and impaired limb use, without changing tactile hypersensitivity, and was fully reversed by the KOR antagonist nor-binaltorphimine. U50,488 treatment was higher in efficacy and duration of action at later time points. U50,488 blocked this pain without altering tumor-induced bone loss or tumor growth. Follow-up studies in human cancer cell lines confirmed that KOR agonists do not affect cancer cell proliferation. These studies suggest that KOR agonists could be a new target for cancer pain management that does not induce cancer cell proliferation or alter bone loss. PERSPECTIVE: This study demonstrates the efficacy of KOR agonists in the treatment of bone cancer-induced pain in mice, without changing tumor size or proliferation in cancer cell lines. This suggests that KOR agonists could be used to manage cancer pain without the drawbacks of mu opioid agonists and without worsening disease progression.