Sustained metabolic benefits of ΔTRTX-Ac1, a tarantula venom-derived peptide, when administered together with exenatide in high-fat fed mice.

AIM: The aim of the present study was to assess the long-term therapeutic efficacy of a recently discovered 28 amino acid peptide, Δ-theraphotoxin-Ac1 (Δ-TRTX-Ac1), originally isolated from venom of the Aphonopelma chalcodes tarantula. Δ-TRTX-Ac has previously been shown to improve pancreatic beta-cell function and suppress appetite. MATERIALS AND METHODS: Δ-TRTX-Ac1 was administered twice daily in high-fat fed (HFF) mice with streptozotocin (STZ)-induced insulin deficiency, namely HFF/STZ mice, for 28 days both alone and in combination with the venom-derived glucagon-like peptide-1 (GLP-1) mimetic, exenatide. RESULTS: Initial pharmacokinetic profiling of ΔTRTX-Ac1 revealed a plasma half-life of 2 h in mice, with ΔTRTX-Ac1 also evidenced in the pancreas 12 h post-injection. Accordingly, HFF-STZ mice received twice-daily injections of Δ-TRTX-Ac1, exenatide or a combination of both peptides for 28 days. As anticipated, HFF/STZ mice presented with hyperglycaemia, impaired glucose tolerance, decreased plasma and pancreatic insulin and disturbed pancreatic islet morphology. Administration of ΔTRTX-Ac1 reduced body weight, improved glucose tolerance and augmented pancreatic insulin content while decreasing glucagon content. Exenatide had similar benefits on body weight and pancreatic hormone content while also reducing circulating glucose. ΔTRTX-Ac1 decreased energy expenditure on day 28 whereas exenatide had no impact. All treatment regimens restored pancreatic islet and beta-cell area towards lean control levels, which was linked to significantly elevated beta-cell proliferation rates. In terms of benefits of combined ΔTRTX-Ac1 and exenatide treatment over individual agents, there was augmentation of glucose tolerance and ambulatory activity with combination therapy, and these mice presented with increased pancreatic glucagon. CONCLUSION: These data highlight the therapeutic promise of ΔTRTX-Ac1 for diabetes, with suggestion that benefits could be enhanced through combined administration with exenatide.

A Cross-Sectional Study of Myopia and Morning Melatonin Status in Northern Irish Adolescent Children.

Purpose: Previous studies have demonstrated an association between melatonin status and both refractive error and axial length in young adult myopes. This study aimed to determine if this relationship extends to a younger adolescent cohort. Methods: Healthy children aged 12-15 years provided morning saliva samples before attending Ulster University (55°N) for cycloplegic autorefraction and axial length measures. Participants completed questionnaires describing recent sleep habits and physical activity. Salivary melatonin was quantified using high-performance liquid chromatography-tandem mass spectrometry. Data collection for all participants occurred over a 1-week period (April 2021). Results: Seventy participants aged 14.3 (95% CI: 14.2-14.5) years were categorised by spherical equivalent refraction [SER] (range: -5.38DS to +1.88DS) into two groups; myopic SER ≤ -0.50DS (n = 22) or nonmyopic -0.50DS < SER ≤ +2.00DS (n = 48). Median morning salivary melatonin levels were 4.52 pg/ml (95% CI: 2.60-6.02) and 4.89 pg/ml (95% CI: 3.18-5.66) for myopic and nonmyopic subjects, respectively, and did not differ significantly between refractive groups (P = 0.91). Melatonin levels were not significantly correlated with SER, axial length, sleep, or activity scores (Spearman's rank, all P > 0.39). Higher levels of physical activity were associated with higher sleep quality (Spearman's rank, ρ = -0.28, P = 0.02). Conclusion: The present study found no significant relationship between morning salivary melatonin levels and refractive error or axial length in young adolescents. This contrasts with outcomes from a previous study of adults with comparable methodology, season of data collection, and geographical location. Prospective studies are needed to understand the discrepancies between adult and childhood findings and evaluate whether melatonin levels in childhood are indicative of an increased risk for future onset of myopia and/or faster axial growth trajectories and myopia progression in established myopes. Future work should opt for a comprehensive dim-light melatonin onset protocol to determine circadian phase.

Successful Proof-of-Concept for Topical Delivery of Novel Peptide ALM201 with Potential Usefulness for Treating Neovascular Eye Disorders.

Purpose: To evaluate the therapeutic benefit of a novel peptide, ALM201, in ocular pathologic vascularization. Design: Experimental study in mouse, rat, and rabbit animal models. Participants: Ten-week-old Lister Hooded male rats, 8-week-old Brown Norway male rats, 9-day-old C57BL/6J mice, and 12-month-old New Zealand male rabbits. Methods: Corneal vascularization was scored for vessel density and vessel distance to suture in a rat corneal suture model. Ocular penetration and biodistribution were evaluated by matrix-assisted laser desorption/ionization mass spectrometry imaging after topical ALM201 application to rabbit eyes. A mouse choroidal sprouting assay, with aflibercept as positive control, was used to evaluate choroidal neovascularization (CNV) in the posterior segment tissue. Efficacy of topical ALM201 was assessed using a rat laser CNV model of neovascular age-related macular degeneration. Main Outcome Measures: Clinical scoring and histologic analysis of vascularized corneas, sprouting area, lesion size, and vessel leakiness in posterior segments. Results: Assessment of ALM201 treatment in the rat corneal suture model showed a significant decrease in vessel density (P = 0.0065) and vessel distance to suture (P = 0.021) compared with vehicle control (phosphate-buffered saline [PBS]). Infiltration of inflammatory cells into the corneal stroma also was reduced significantly compared with PBS (724.5 ± 122 cells/mm2 vs. 1837 ± 195.9 cells/mm2, respectively; P = 0.0029). Biodistribution in rabbit eyes confirmed ALM201 bioavailability in anterior and posterior ocular segments 1 hour after topical instillation. ALM201 treatment significantly suppressed choroid vessel sprouting when compared with PBS treatment (44.5 ± 14.31 pixels vs. 120.9 ± 33.37 pixels, respectively; P = 0.04) and was not inferior to aflibercept (65.63 ± 11.86 pixels; P = 0.7459). Furthermore, topical ALM201 significantly improved vessel leakiness (leakage scores: 2.1 ± 0.7 vs. 2.9 ± 0.1; P = 0.0274) and lesion size (144,729 ± 33,239 µm3 vs. 187,923 ± 28,575 µm3; P = 0.03) in the rat laser CNV model when compared with topical PBS vehicle. Conclusions: ALM201 is a promising novel molecule with anti-inflammatory and antivascularization activity and is a strong candidate to meet the clinical need of a new, topically delivered therapeutic agent for treating inflammation and pathologic vascularization in the anterior and posterior segments of the eye.

Vitamin D and Bone Health of Older Adults within Care Homes: An Observational Study.

Limited studies have reported vitamin D status and health outcomes in care home residents, a group at risk of vitamin D deficiency. This study investigated serum 25-hydroxyvitamin D (25-OHD) concentrations in older adults within care homes in Northern Ireland (NI) and its association with musculoskeletal health (ultrasound T-score, muscle strength, Timed Up & Go test (TUG)), bone turnover markers (BTMs), and immune function markers. A total of 87 participants were recruited with mean ± SD age 83.2 ± 7.9 years. Mean ± SD serum 25-OHD concentration (n 69) was 49.52 ± 35.58 nmol/L. Vitamin D deficiency (25-OHD <25 nmol/L) was observed in 34.8% (n 24) of participants with 17.4% (n 12) classified as insufficient (25-OHD 25−50 nmol/L) and 47.8% (n 33) as sufficient (25-OHD >50 nmol/L). 25-OHD concentration was not an independent predictor of T-score, muscle strength, TUG, or inflammatory cytokines. After adjusting for covariates, a significant negative association was observed between 25-OHD concentration and the BTMs; osteocalcin (ß = −0.395; p = 0.001), procollagen type 1 N propeptide (P1NP) (ß = −0.320; p = 0.012), and C-terminal telopeptide of type 1 collagen (CTX) (ß = −0.377; p = 0.003). Higher 25-OHD concentration was positively associated with use of vitamin D ± calcium supplementation (ß = 0.610; p < 0.001). Vitamin D deficiency and insufficiency were highly prevalent in this sample of care home residents in NI. Higher 25-OHD concentration was associated with greater supplement use and with reduced bone turnover, which in this population is linked with reduced bone loss. These findings emphasize the need for a mandatory vitamin D ± calcium supplementation policy specific for care home residents.

Biomarkers for Detecting Kidney Dysfunction in Type-2 Diabetics and Diabetic Nephropathy Subjects: A Case-Control Study to Identify Potential Biomarkers of DN to Stratify Risk of Progression in T2D Patients.

Introduction: Currently there are no biomarkers that are predictive of when patients with type-2 diabetes (T2D) will progress to more serious kidney disease i.e., diabetic nephropathy (DN). Biomarkers that could identify patients at risk of progression would allow earlier, more aggressive treatment intervention and management, reducing patient morbidity and mortality. Materials and Methods: Study participants (N=88; control n=26; T2D n=32; DN n=30) were recruited from the renal unit at Antrim Area Hospital, Antrim, UK; Whiteabbey Hospital Diabetic Clinic, Newtownabbey, UK; Ulster University (UU), Belfast, UK; and the University of the Third Age (U3A), Belfast, UK; between 2019 and 2020. Venous blood and urine were collected with a detailed clinical history for each study participant. Results: In total, 13/25 (52.0%) biomarkers measured in urine and 25/34 (73.5%) biomarkers measured in serum were identified as significantly different between control, T2D and DN participants. DN patients, were older, smoked more, had higher systolic blood pressure and higher serum creatinine levels and lower eGFR function. Serum biomarkers significantly inversely correlated with eGFR. Conclusion: This pilot-study identified several serum biomarkers that could be used to predict progression of T2D to more serious kidney disease: namely, midkine, sTNFR1 and 2, H-FABP and Cystatin C. Our results warrant confirmation in a longitudinal study using a larger patient cohort.

Spatial localization of ß-unsaturated aldehyde markers in murine diabetic kidney tissue by mass spectrometry imaging.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Limitations in current diagnosis and screening methods have sparked a search for more specific and conclusive biomarkers. Hyperglycemic conditions generate a plethora of harmful molecules in circulation and within tissues. Oxidative stress generates reactive α-dicarbonyls and ß-unsaturated hydroxyhexenals, which react with proteins to form advanced glycation end products. Mass spectrometry imaging (MSI) enables the detection and spatial localization of molecules in biological tissue sections. Here, for the first time, the localization and semiquantitative analysis of "reactive aldehydes" (RAs) 4-hydroxyhexenal (4-HHE), 4-hydroxynonenal (4-HNE), and 4-oxo-2-nonenal (4-ONE) in the kidney tissues of a diabetic mouse model is presented. Ionization efficiency was enhanced through on-tissue chemical derivatization (OTCD) using Girard's reagent T (GT), forming positively charged hydrazone derivatives. MSI analysis was performed using matrix-assisted laser desorption ionization (MALDI) coupled with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR). RA levels were elevated in diabetic kidney tissues compared to lean controls and localized throughout the kidney sections at a spatial resolution of 100 µm. This was confirmed by liquid extraction surface analysis-MSI (LESA-MSI) and liquid chromatography-mass spectrometry (LC-MS). This method identified ß-unsaturated aldehydes as "potential" biomarkers of DN and demonstrated the capability of OTCD-MSI for detection and localization of poorly ionizable molecules by adapting existing chemical derivatization methods. Untargeted exploratory distribution analysis of some precursor lipids was also assessed using MALDI-FT-ICR-MSI.

Vitamin D Status and Health Outcomes in School Children in Northern Ireland: Year One Results from the D-VinCHI Study.

(1) Background: Vitamin D status has never been investigated in children in Northern Ireland (UK). (2) Methods: Children (4-11 years) (n = 47) were recruited from November 2019 to March 2020 onto the cross-sectional study. Anthropometry was assessed. Plasma 25-hydroxyvitamin D (25(OH)D) was analysed. Vitamin D intake, parental knowledge and perceptions, participant habits, physical activity and sedentary behaviour were established via questionnaire. Muscle strength was assessed via isometric grip strength dynamometry and balance via dominant single-leg and tandem stance. Parathyroid hormone, bone turnover markers (OC, CTX and P1NP), glycated haemoglobin and inflammatory markers (CRP, IFN-γ, IL-10, IL-12p70, IL-13, IL-1ß, IL-2, IL-4, IL-6, IL-8 and TNF-α) were analysed. (3) Results: Mean (SD) 25(OH)D was 49.17 (17.04) nmol/L (n = 47); 44.7% of the children were vitamin D sufficient (25(OH)D >50 nmol/L), 48.9% were insufficient (25-50 nmol/L) and 6.4% were deficient (<25 nmol/L). 25(OH)D was positively correlated with vitamin D intake (µg/day) (p = 0.012, r = 0.374), spring/summer outdoor hours (p = 0.006, r = 0.402) and dominant grip strength (kg) (p = 0.044, r = 0.317). Vitamin D sufficient participants had higher dietary vitamin D intake (µg/day) (p = 0.021), supplement intake (µg/day) (p = 0.028) and spring/summer outdoor hours (p = 0.015). (4) Conclusion: Over half of the children were vitamin D deficient or insufficient. Wintertime supplementation, the consumption of vitamin D rich foods and spring/summer outdoor activities should be encouraged to minimise the risk of vitamin D inadequacy.

On-tissue chemical derivatization in mass spectrometry imaging.

Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrix-assisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed on-tissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCD-MSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive.

Analysis of reactive aldehydes in urine and plasma of type-2 diabetes mellitus patients through liquid chromatography-mass spectrometry: Reactive aldehydes as potential markers of diabetic nephropathy.

Introduction: Diabetes is a major public health issue that is approaching epidemic proportions globally. Diabetes mortality is increasing in all ethnic groups, irrespective of socio-economic class. Obesity is often seen as the main contributor to an increasing prevalence of diabetes. Oxidative stress has been shown to trigger obesity by stimulating the deposition of white adipose tissue. In this study, we measured reactive aldehydes by liquid chromatography-mass spectrometry (LC-MS), in the urine and plasma of type-2 diabetic mellitus (T2DM) patients, as potential surrogates of oxidative stress. Our hypothesis was that reactive aldehydes play a significant role in the pathophysiology of diabetes, and these reactive species, may present potential drug targets for patient treatment. Materials and methods: Study participants [N = 86; control n = 26; T2DM n = 32, and diabetic nephropathy (DN) n = 28] were recruited between 2019 and 2020. Urine and blood samples were collected from all participants, including a detailed clinical history, to include patient behaviours, medications, and co-morbidities. Reactive aldehyde concentrations in urine and plasma were measured using pre-column derivatisation and LC-MS, for control, T2DM and DN patients. Results: Reactive aldehydes were measured in the urine and plasma of control subjects and patients with T2DM and DN. In all cases, the reactive aldehydes under investigation; 4-HNE, 4-ONE, 4-HHE, pentanal, methylglyoxal, and glyoxal, were significantly elevated in the urine and serum of the patients with T2DM and DN, compared to controls (p < 0.001) (Kruskal-Wallis). Urine and serum reactive aldehydes were significantly correlated (≥0.7) (p < 0.001) (Spearman rho). The concentrations of the reactive aldehydes were significantly higher in plasma samples, when compared to urine, suggesting that plasma is the optimal matrix for screening T2DM and DN patients for oxidative stress. Conclusion: Reactive aldehydes are elevated in the urine and plasma of T2DM and DN patients. Reactive aldehydes have been implicated in the pathobiology of T2DM. Therefore, if reactive aldehydes are surrogates of oxidative stress, these reactive aldehyde species could be therapeutic targets for potential drug development.

Derivatization with 2-hydrazino-1-methylpyridine enhances sensitivity of analysis of 5α-dihydrotestosterone in human plasma by liquid chromatography tandem mass spectrometry.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of DHT by LC-MS/MS. Derivatization of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of DHT, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies.

Maternal and child fatty acid desaturase genotype as determinants of cord blood long-chain PUFA (LCPUFA) concentrations in the Seychelles Child Development Study.

Optimal maternal long-chain PUFA (LCPUFA) status is essential for the developing fetus. The fatty acid desaturase (FADS) genes are involved in the endogenous synthesis of LCPUFA. The minor allele of various FADS SNP have been associated with increased maternal concentrations of the precursors linoleic acid (LA) and α-linolenic acid (ALA), and lower concentrations of arachidonic acid (AA) and DHA. There is limited research on the influence of FADS genotype on cord PUFA status. The current study investigated the influence of maternal and child genetic variation in FADS genotype on cord blood PUFA status in a high fish-eating cohort. Cord blood samples (n 1088) collected from the Seychelles Child Development Study (SCDS) Nutrition Cohort 2 (NC2) were analysed for total serum PUFA. Of those with cord PUFA data available, maternal (n 1062) and child (n 916), FADS1 (rs174537 and rs174561), FADS2 (rs174575), and FADS1-FADS2 (rs3834458) were determined. Regression analysis determined that maternal minor allele homozygosity was associated with lower cord blood concentrations of DHA and the sum of EPA + DHA. Lower cord blood AA concentrations were observed in children who were minor allele homozygous for rs3834458 (ß = 0·075; P = 0·037). Children who were minor allele carriers for rs174537, rs174561, rs174575 and rs3834458 had a lower cord blood AA:LA ratio (P < 0·05 for all). Both maternal and child FADS genotype were associated with cord LCPUFA concentrations, and therefore, the influence of FADS genotype was observed despite the high intake of preformed dietary LCPUFA from fish in this population.

Spatial distribution of isobaric androgens in target tissues using chemical derivatization and MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumour regression, but commonly restricted by the eventual emergence of a more lethal 'castrate resistant' (CRPC) form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). Decoding this tissue-specific metabolic pathway is key for the development of novel therapeutic treatments. Mass spectrometry imaging (MSI) is an analytical technique that allows the visualization of the distribution of numerous classes of biomolecules within tissue sections. The analysis of androgens by liquid chromatography mass spectrometry (LC/MS)-based methods however presents a challenge due to their generally poor ionization efficiency and low physiological endogenous levels. In MSI, on-tissue chemical derivatization (OTCD) has enabled the limits of steroids to be imaged within tissues to be pushed by overcoming poor ionization performance. However, isobaric interference of key androgen derivatives such as T and DHEA can severely hamper studying the intracrinology in several diseases. Here, we have evaluated the use of laser induced post-ionization (MALDI-2) combined with trapped ion mobility separation (TIMS) and orthogonal time-of-flight (QTOF) MS for the visualization of isobaric derivatized androgens in murine tumour xenograft at about 50 µm spatial resolution. With this combination, isobaric T and DHEA were separated in tissue sections and the signals of derivatized steroids enhanced by about 20 times. The combination of TIMS and MALDI-2 thus shows unique potential to study tissue intracrinology within target tissues. This could offer the opportunity for many novel insights into tissue-specific androgen biology.

The influence of fish consumption on serum n-3 polyunsaturated fatty acid (PUFA) concentrations in women of childbearing age: a randomised controlled trial (the iFish Study).

PURPOSE: Long-chain polyunsaturated fatty acids (LCPUFA) can be synthesised endogenously from linoleic acid (LA) and α-linolenic acid (ALA) in a pathway involving the fatty acid desaturase (FADS) genes. Endogenous synthesis is inefficient; therefore, dietary intake of preformed LCPUFA from their richest source of fish is preferred. This study investigated the effect of fish consumption on PUFA concentrations in women of childbearing age while stratifying by FADS genotype. The influence of fish consumption on lipid profile, and markers of inflammation and oxidative stress was also examined. METHODS: Healthy women (n = 49) provided a buccal swab which was analysed for FADS2 genotype (rs3834458; T/deletion). Participants were stratified according to genotype and randomised to an intervention group to receive either no fish (n = 18), 1 portion (n = 14) or 2 portions (n = 17) (140 g per portion) of fish per week for a period of 8 weeks. Serum PUFA was analysed at baseline and post-intervention. Lipid profile, and markers of inflammation and oxidative stress were also analysed. RESULTS: Participants consuming 2 portions of fish per week had significantly higher concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 PUFA, and a lower n-6:n-3 ratio compared to those in the no fish or 1 portion per week group (all p < 0.05). Fish consumption did not have a significant effect on biomarkers of oxidative stress, inflammation and lipid profile in the current study. CONCLUSION: Consumption of 2 portions of fish per week has beneficial effects on biological n-3 PUFA concentrations in women of childbearing age; however, no effects on oxidative stress, inflammation or lipid profile were observed. This trial was registered at www.clinicaltrials.gov (NCT03765580), registered December 2018.

Clinical and Laboratory Associations with Methotrexate Metabolism Gene Polymorphisms in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that causes loss of joint function and significantly reduces quality of life. Plasma metabolite concentrations of disease-modifying anti-rheumatic drugs (DMARDs) can influence treatment efficacy and toxicity. This study explored the relationship between DMARD-metabolising gene variants and plasma metabolite levels in RA patients. DMARD metabolite concentrations were determined by tandem mass-spectrometry in plasma samples from 100 RA patients with actively flaring disease collected at two intervals. Taqman probes were used to discriminate single-nucleotide polymorphism (SNP) genotypes in cohort genomic DNA: rs246240 (ABCC1), rs1476413 (MTHFR), rs2231142 (ABCG2), rs3740065 (ABCC2), rs4149081 (SLCO1B1), rs4846051 (MTHFR), rs10280623 (ABCB1), rs16853826 (ATIC), rs17421511 (MTHFR) and rs717620 (ABCC2). Mean plasma concentrations of methotrexate (MTX) and MTX-7-OH metabolites were higher (p < 0.05) at baseline in rs4149081 GA genotype patients. Patients with rs1476413 SNP TT or CT alleles have significantly higher (p < 0.001) plasma poly-glutamate metabolites at both study time points and correspondingly elevated disease activity scores. Patients with the rs17421511 SNP AA allele reported significantly lower pain scores (p < 0.05) at both study intervals. Genotyping strategies could help prioritise treatments to RA patients most likely to gain clinical benefit whilst minimizing toxicity.

Sleep, a Governor of Morbidity in PTSD: A Systematic Review of Biological Markers in PTSD-Related Sleep Disturbances.

BACKGROUND: Sleep disturbances (SD) are the most impactful and commonly reported symptoms in post-traumatic stress disorder (PTSD). Yet, they are often resistant to primary PTSD therapies. Research has identified two distinct SDs highly prevalent in PTSD; insomnia and nightmares. Those who report SDs prior to a traumatic event are at greater risk for developing PTSD; highlighting that sleep potentially plays a role in PTSD's pathology. To further understand the pathobiological mechanisms that lead to the development of PTSD, it is first imperative to understand the interplay which exists between sleep and PTSD on a biological level. The aim of this systematic review is to determine if biological or physiological markers are related to SD in PTSD. METHODS: A systematic literature search was conducted on the electronic databases; Medline, Embase, AMED and PsycINFO, using Medical Subject Headings and associated keywords. RESULTS: Sixteen studies were included in the final analyses. Physiological makers of autonomic function, and biochemical markers of HPA-axis activity; inflammatory processes; and trophic factor regulation were related to the severity of SDs in PTSD. CONCLUSION: These findings add to the growing literature base supporting a central focus on sleep in research aiming to define the pathophysiological processes which result in PTSD, as well as emphasising the importance of specifically targeting sleep as part of a successful PTSD intervention strategy. Resolving SDs will not only reduce PTSD symptom severity and improve quality of life but will also reduce all-cause mortality, hospital admissions and lifetime healthcare costs for those with PTSD. Limitations of the current literature are discussed, and key recommendations future research must adhere to are made within.

Elevated Melatonin Levels Found in Young Myopic Adults Are Not Attributable to a Shift in Circadian Phase.

Purpose: To evaluate the relationship between refractive error, circadian phase, and melatonin with consideration of prior light exposure, physical activity, and sleep. Methods: Healthy young myopic (spherical equivalent refraction [SER] ≤-0.50DS) and emmetropic adults underwent noncycloplegic autorefraction and axial length (AL) measures. Objective measurements of light exposure, physical activity, and sleep were captured across 7 days by wrist-worn Actiwatch-2 devices. Questionnaires assessed sleep quality and chronotype. Hourly evening saliva sampling during a dim-light melatonin onset (DLMO) protocol evaluated circadian phase, and both morning serum and saliva samples were collected. Liquid chromatography/mass spectrometry quantified melatonin. Results: Subjects (n = 51) were aged 21.4 (interquartile range, 20.1-24.0) years. Melatonin was significantly higher in the myopic group at every evening time point and with both morning serum and saliva sampling (P ≤ 0.001 for all). DLMO-derived circadian phase did not differ between groups (P = 0.98). Multiple linear regression analysis demonstrated significant associations between serum melatonin and SER (B = -.34, ß = -.42, P = 0.001), moderate activity (B = .009, ß = .32, P = 0.01), and mesopic illumination (B = -.007, ß = -.29, P = 0.02), F(3, 46) = 7.23, P < 0.001, R2 = 0.32, R2adjusted = .28. Myopes spent significantly more time exposed to "indoor" photopic illumination (3 to ≤1000 lux; P = 0.05), but "indoor" photopic illumination was not associated with SER, AL, or melatonin, and neither sleep, physical activity, nor any other light exposure metric differed significantly between groups (P > 0.05 for all). Conclusions: While circadian phase is aligned in adult myopes and emmetropes, myopia is associated with both elevated serum and salivary melatonin levels. Prospective studies are required to ascertain whether elevated melatonin levels occur before, during, or after myopia development.

Spatial Localization of Vitamin D Metabolites in Mouse Kidney by Mass Spectrometry Imaging.

Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD-MALDI-MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD-MSI by adapting existing CD methods.

Non-photopic and photopic visual cycles differentially regulate immediate, early, and late phases of cone photoreceptor-mediated vision.

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters to 11-cis-retinol is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early, and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, while the emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaptation displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 min of light, early photopic vision was recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the nonphotopic or photopic visual cycles for mediating vision in bright light.

Assessing the Oxidative Degradation of N-Methylpyrrolidone (NMP) in Microelectronic Fabrication Processes by Using a Multiplatform Analytical Approach.

During the construction of recording head devices, corrosion of metal features and subsequent deposition of corrosion by-products have been observed. Previous studies have determined that the use of N-methylpyrrolidone (NMP) may be a contributing factor. In this study, we report the use of a novel multiplatform analytical approach comprising of pH, liquid chromatography/UV detection (LC/UV), inductively coupled plasma optical emission spectroscopy (ICP-OES), and LC/mass spectrometry (LC/MS) to demonstrate that reaction conditions mimicking those of general photoresist removal processes can invoke the oxidation of NMP during the photolithography lift-off process. For the first time, we have confirmed that the oxidation of NMP lowers the pH, facilitating the dissolution of transition metals deposited on wafer substrates during post-mask and pre-lift-off processes in microelectronic fabrication. This negatively impacts upon the performance of the microelectronic device. Furthermore, it was shown that, by performing the process in an inert atmosphere, the oxidation of NMP was suppressed and the pH was stabilized, suggesting an affordable modification of the photolithography lift-off stage to enhance the quality of recording heads. This novel study has provided key data that may have a significant impact on current and future fabrication process design, optimization, and control. Results here suggest the inclusion of pH as a key process input variable (KPIV) during the design of new photoresist removal processes.

Effects of vitamin D as a regulator of androgen intracrinology in LNCAP prostate cancer cells.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumor regression, but commonly restricted by the eventual emergence of a more lethal 'castrate resistant' form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids, are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). In this study, we explored the capacity of the active vitamin D hormone to interact and elicit changes upon this prostatic intracrine pathway at a metabolic level. We used androgen dependent LNCaP cells cultured under steroid-depleted conditions and assessed the impact of vitamin D-based compounds upon intracrine pathways that convert exogenously added DHEA to relevant metabolites, through Mass Spectrometry (MS). Changes in relevant metabolism-related gene targets were also assessed. Our findings confirm that exposure to vitamin D based compounds, within LNCaP cells, elicits measurable and significant reduction in the intracrine conversion of DHEA to T, DHT and other intermediate metabolites within the androgenic pathway. The aassessment and validation of the biological model and analytical platforms were performed by pharmacological manipulations of the SRD5α and HSD-17ß enzymes. The data provides further confirmation for how a vitamin D-based regime may be used to counter intracrine mechanisms contributing to the emergence of castrate-resistant tumors.