Molecular Pathway, Epidemiological Data and Treatment Strategies of Fungal Infection (Mycoses): A Comprehensive Review.

The recent increase in fungal infections is a health crisis. This surge is directly tied to the increase in immunocompromised people caused by changes in medical practice, such as the use of harsh chemotherapy and immunosuppressive medicines. Immunosuppressive disorders such as HIV have exacerbated the situation dramatically. Subcutaneous or superficial fungal infections can harm the skin, keratinous tissues, and mucous membranes. This category includes some of the most common skin disorders that impact millions of people worldwide. Despite the fact that they are seldom fatal, they can have a catastrophic impact on a person's quality of life and, in rare situations, spread to other people or become obtrusive. The majority of fungal infections under the skin and on the surface are simply and quickly cured. An opportunistic organism that preys on a weak host or a natural intruder can both result in systemic fungal infections. Furthermore, it might be exceedingly lethal and dangerous to one's life. Dimorphic fungi may pose a hazard to healthy populations that are not exposed to endemic fungi. Increased surveillance, the availability of quick, noninvasive diagnostic tests, monitoring the emergence of antifungal medication resistance, and research on the pathophysiology, prevention, and management of fungal infections are just a few potential solutions to these new health problems. The goal of this review is to summarize the data available for fungal infections and the different therapies which are involved in their treatment. Additionally, it also summarizes the molecular and scientific data of the plants which contain anti-fungal activity. Data are acquired using Google, PubMed, Scholar, and other online sources.

Evaluating the use of locked nucleic acid capture probes in hybrid LC-MS/MS analysis of siRNA analytes.

Background: Hybrid LC-MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC-MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNA compounds as model analytes, hybrid LC-MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for siRNA analytes.

Method development workflow for quantifying protein biomarkers by hybrid LC-MS/MS.

Background: Industry-standard guidance on method development and validation of hybrid LC-MS/MS assays for protein biomarkers, particularly on evaluation of parallelism, is lacking. Methods: Using a protein endogenous to humans and mice as a model analyte, a quantitative hybrid LC-MS/MS workflow was developed using a surrogate matrix approach with a recombinant form of the protein as the calibrant. Results: The developed workflow identified a surrogate matrix, established parallelism between the surrogate and authentic matrices and assessed parallelism between the recombinant and authentic forms of the protein. The final method was qualified using precision and accuracy with recovery assessments. Conclusion: The established workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for endogenous protein biomarkers.

Multiassay nutritional metabolomics profiling of low vitamin A status versus adequacy is characterized by reduced plasma lipid mediators among lactating women in the Philippines: A pilot study.

Low vitamin A (VA) status is common among lactating women in low-income countries. Lactation has substantial effects on mother's metabolism and VA is required in multiple biological processes, including growth, vision, immunity, and reproduction. The objective of this pilot study was to use metabolomics profiling to conduct a broad, exploratory assessment of differences in plasma metabolites associated with low VA status versus VA adequacy in lactating women. Plasma samples from lactating women who participated in a survey in Samar, Philippines, were selected from a cross-sectional study based on plasma retinol concentrations indicating low (VA-; n = 5) or adequate (VA+; n = 5) VA status (plasma retinol <0.8 or >1.05 µmol/L). The plasma results collected from 6 metabolomics assays (oxylipins, endocannabinoids, bile acids, primary metabolomics, biogenic amines, and lipidomics) were compared by group using liquid chromatography mass spectrometry. Twenty-eight metabolites were altered in the VA- versus VA+ status groups, with 24 being lipid mediators (P < .05). These lipid mediators included lower concentrations of arachidonic acid- and eicosapentaenoic acid-derived oxylipins, as well as lysophospholipids and sphingolipids, in the VA- group (P < .05). Chemical similarity enrichment analysis identified hydroxy-eicosatetraenoic acids, hydroxy-eicosapentaenoic acids, and dihydroxy-eicosatetraenoic acids as significantly altered oxylipin clusters (P < .0001, false discovery rate [FDR] P < .0001), as well as sphingomyelins, saturated lysophosphatidylcholines, phosphatidylcholines, and phosphatidylethanolamines (P < .001, FDR P < .01). The multiassay nutritional metabolomics profiling of low VA status compared with adequacy in lactating women was characterized by reduced lipid mediator concentrations. Future studies with stronger study designs and larger sample size are needed to confirm and validate these preliminary results.

A validated surrogate analyte LC-MS/MS assay for quantification of endogenous cortisol in human whole blood.

Cortisol is a steroid hormone that is frequently measured as a marker of stress, inflammation, and immune function. While commonly analyzed in saliva, hair, blood plasma and urine, a recent trend towards whole blood-based at-home collection devices has emerged, which necessitates development of more sensitive assays for cortisol in whole blood. To support the implementation of a patient-centric sampling approach in a drug development program, a fit-for-purpose surrogate analyte-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for cortisol in whole blood was developed using 13C3-cortisol as a surrogate analyte and cortisol-d6 as the internal standard. The surrogate analyte approach was chosen due to a lack of available cortisol-free whole blood and the absence of appropriately representative surrogate matrices. Samples were prepared using supported liquid extraction, and the LC-MS/MS analysis consisted of a 4.00 min analytical run. The method demonstrated linearity between 0.500 and 500 ng/mL of 13C3-cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and 13C3-cortisol-based quality control (QC) samples when quantified against a 13C3-cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a 13C3-cortisol-based calibration curve also suggests parallelism between the analytes. These results indicate a viable surrogate analyte method, that is fit-for-purpose to analyze whole blood cortisol levels using a surrogate analyte LC-MS/MS approach. Evaluation of patient samples showed very promising comparability between whole blood and plasma cortisol concentrations, suggesting that whole blood could be used in place of or in addition to a plasma-based sampling protocol in clinical trials analyzing cortisol. Overall, this method presents a novel tool that is a first step in supporting the trend towards sample miniaturization and at-home sample collection, and may be readily used in clinical and diagnostic settings.

Quantification of the anti-murine PD-1 monoclonal antibody RMP1-14 in BALB/c mouse plasma by liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study.

RMP1-14 is a monoclonal antibody that targets the murine PD-1 protein, and has been used extensively to probe the effects of PD-1 inhibition in preclinical murine models. However, to date, no quantitative analytical methods have been published for RMP1-14. To evaluate its anti-tumor activity in BALB/c mice inoculated with CT26.WT murine colon cancer cells, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify RMP1-14 in BALB/c mouse K3EDTA plasma was developed and validated. The methodology used a signature peptide (GFYPPDIYTEWK) as a surrogate for RMP1-14 quantitation and an isotopically labeled analog of the signature peptide as the internal standard. Initial method development focused on a hybrid LC-MS/MS assay involving Protein G immunoprecipitation, but this strategy was abandoned due to lack of selectivity. The final validated method consisted of dilution with Tris-buffered saline, trypsin digestion, and desalting using micro solid-phase extraction. Analytical run time was 3.50 min, and the method demonstrated linearity between 0.500 and 50.0 µg/mL of intact RMP1-14. Accuracy, precision, and robustness were all acceptable, and the method was demonstrated to be comparable to a commercially available fit-for-purpose enzyme-linked immunosorbent assay (ELISA) capable of measuring RMP1-14. The validated method was used to generate pharmacokinetic parameters from tumor-bearing BALB/c mice dosed with RMP1-14 at either 2.50 or 7.50 mg/kg. Overall, the validated method represents a novel tool that can be used to evaluate RMP1-14 activity in future immuno-oncology studies.

Noninvasive profiling of sweat-derived lipid mediators for cutaneous research.

BACKGROUND AND OBJECTIVE: Recent increases in the use of noninvasive matrices for biomedical analysis has led to interest in the evaluation of sweat for both clinical and research applications. However, despite being one of the two main cutaneous secretions, until very recently, only one study actually analyzed sweat in the context of cutaneous disease. This review attempts to make the case for increased use of sweat in cutaneous research, and discusses lipid mediators as potential analytical targets in sweat. METHODS: Sweat composition and its relationship with the skin and systemic circulation are discussed, as are practical considerations for sweat sampling and analysis. Previous analyses of lipid mediators in skin biopsies are provided to show that lipid mediators can regulate cutaneous processes and disease pathways. Summaries of recent studies involving the analysis of sweat lipid mediators are provided to demonstrate the utility of sweat lipid mediator testing to support future cutaneous research studies. RESULTS: Sweat has the potential to reflect both local and systemic biochemical changes in response to disease or intervention, and two recent studies of sweat lipid mediators confirm this ability. Additionally, sweat lipid mediators appear to be temporally stable with individual variability comparable to other matrices, suggesting that these analytes could be useful biomarkers. CONCLUSIONS: Sweat metabolites may be capable of reporting changes in cutaneous biochemical pathways, thereby providing insight into the immunomodulatory biochemistry of the skin. Lipid mediator analysis of sweat appears to be a non invasive approach that could enhance existing cutaneous research and diagnostic methodologies.

Effects of atopic dermatitis and gender on sebum lipid mediator and fatty acid profiles.

Skin disease alters cutaneous lipid mediator metabolism, and if skin secretions contain evidence of these changes, they may constitute useful clinical matrices with low associated subject burden. The influences of skin diseases on sebum lipid mediators are understudied. Here, sebum oxylipins, endocannabinoids, sphingolipids, and fatty acids were quantified from the non-lesional bilateral cheeks of subjects with and without quiescent atopic dermatitis (AD) using LC-MS/MS and GC-MS. AD decreased C36 [NS] and [NdS] ceramide concentrations. Compared to males, females demonstrated increased concentrations of oxylipin alcohols and ketones, and saturated and monounsaturated non-esterified fatty acids, as well as decreased concentrations of C42 [NS] and [NdS] ceramides. Additionally, contemporaneously collected sweat lipid mediator profiles were distinct, with sebum showing higher concentrations of most targets, but fewer highly polar lipids. Therefore, AD and gender appear to alter sebum lipid metabolism even in non-lesional skin of quiescent subjects.

Oral ibuprofen differentially affects plasma and sweat lipid mediator profiles in healthy adult males.

Sweat contains a variety of lipid mediators, but whether they originate from the plasma filtrate or from the cutaneous sweat glandular tissues themselves is unknown. To explore this knowledge gap, we collected plasma and sweat from healthy men (n = 9) immediately before and 0.5, 2 and 4 h after oral administration of 400 mg ibuprofen. Of the over 100 lipid mediators assayed by liquid chromatography-tandem mass spectrometry, ∼45 were detected in both plasma and sweat, and 36 were common to both matrices. However, baseline concentrations in each matrix were not correlated and metabolite relative abundances between matrices differed. Oral ibuprofen administration altered sweat lipid mediators, reducing prostaglandin E2, linoleoylethanolamide, and oleoylethanolamide, while increasing 11-hydroxyeicosatetraenoic acid, and causing transient changes in 9-nitrooleate, N-arachidonylglycine and 20-hydroxyeicosatetraenoic acid. Meanwhile, plasma N-acylethanolamide concentrations increased with ibuprofen administration. These results suggest that sweat and plasma differentially reflect biochemical changes due to oral ibuprofen administration, and that plasma is unlikely to be the predominant source of the sweat lipid mediator profile.

Effects of stimulation technique, anatomical region, and time on human sweat lipid mediator profiles.

Few studies compare sampling protocol effect on sweat composition. Here we evaluate the impact of sweat stimulation mode and site of collection on lipid mediator composition. Sweat from healthy males (n=7) was collected weekly for three weeks from the volar forearm following either pilocarpine iontophoresis or exercise, and from the forearm, back and thigh following pilocarpine iontophoresis only. Sweat content of over 150 lipid mediators were measured by liquid chromatography-tandem mass spectrometry. Seventy lipid mediators were routinely detected, including prostanoids, alcohols, diols, epoxides, ketones, nitrolipids, N-acylethanolamides, monoacylglycerols, and ceramides. Detected lipid mediators appeared unaffected by sampling site, though the forearm was the most consistent source of sweat. Pilocarpine-induced sweat showed increased concentrations of most detected compounds. Moreover, lipid mediator concentrations and profiles were temporally stable over the study duration. Sweat therefore appears to be a consistent and anatomically-stable source of lipid mediators, but care must be taken in comparing results obtained from different stimulation techniques.

Oleocanthal-rich extra virgin olive oil demonstrates acute anti-platelet effects in healthy men in a randomized trial.

The phenolic profiles of extra virgin olive oils (EVOOs) may influence their cardiovascular benefits. In a randomized crossover of acute EVOO intake on platelet function, participants (n=9) consumed 40 mL of EVOO weekly. EVOOs were matched for total phenolic content and were either tyrosol-poor with 1:2 oleacein/oleocanthal (D2i0.5), or 2:1 oleacein/oleocanthal (D2i2), or predominantly tyrosol (D2i0). Ibuprofen provided a platelet inhibition control. Blood was collected pre- and 2 hr post-EVOO intake. D2i0.5 and D2i2 reduced 1 µg/mL collagen-stimulated maximum platelet aggregation (Pmax), with effects best correlated to oleocanthal intake (R=0.56, P=0.002). Total phenolic intake was independently correlated to eicosanoid production inhibition, suggesting that cyclooxygenase blockade was not responsible for the Pmax inhibition. Five participants exhibited >25% ΔPmax declines with D2i0.5 and D2i2 intake and plasma metabolomic profiles discriminated subjects by oil responsivity. Platelet responses to acute EVOO intake are associated with oil phenolic composition and may be influenced by diet.

Sweat lipid mediator profiling: a noninvasive approach for cutaneous research.

Recent advances in analytical and sweat collection techniques provide new opportunities to identify noninvasive biomarkers for the study of skin inflammation and repair. This study aims to characterize the lipid mediator profile including oxygenated lipids, endocannabinoids, and ceramides/sphingoid bases in sweat and identify differences in these profiles between sweat collected from nonlesional sites on the unflared volar forearm of subjects with and without atopic dermatitis (AD). Adapting routine procedures developed for plasma analysis, over 100 lipid mediators were profiled using LC-MS/MS and 58 lipid mediators were detected in sweat. Lipid mediator concentrations were not affected by sampling or storage conditions. Increases in concentrations of C30-C40 [NS] and [NdS] ceramides, and C18:1 sphingosine, were observed in the sweat of study participants with AD despite no differences being observed in transepidermal water loss between study groups, and this effect was strongest in men (P < 0.05, one-way ANOVA with Tukey's post hoc HSD). No differences in oxylipins and endocannabinoids were observed between study groups. Sweat mediator profiling may therefore provide a noninvasive diagnostic for AD prior to the presentation of clinical signs.

A Randomized Placebo Controlled Trial of Ibuprofen for Respiratory Syncytial Virus Infection in a Bovine Model.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and hospital admission in infants. An analogous disease occurs in cattle and costs US agriculture a billion dollars a year. RSV causes much of its morbidity indirectly via adverse effects of the host response to the virus. RSV is accompanied by elevated prostaglandin E2 (PGE2) which is followed by neutrophil led inflammation in the lung. Ibuprofen is a prototypical non-steroidal anti-inflammatory drug that decreases PGE2 levels by inhibiting cyclooxygenase. HYPOTHESES: We hypothesized that treatment of RSV with ibuprofen would decrease PGE2 levels, modulate the immune response, decrease clinical illness, and decrease the histopathological lung changes in a bovine model of RSV. We further hypothesized that viral replication would be unaffected. METHODS: We performed a randomized placebo controlled trial of ibuprofen in 16 outbred Holstein calves that we infected with RSV. We measured clinical scores, cyclooxygenase, lipoxygenase and endocannabinoid products in plasma and mediastinal lymph nodes and interleukin (Il)-4, Il-13, Il-17 and interferon-γ in mediastinal lymph nodes. RSV shedding was measured daily and nasal Il-6, Il-8 and Il-17 every other day. The calves were necropsied on Day 10 post inoculation and histology performed. RESULTS: One calf in the ibuprofen group required euthanasia on Day 8 of infection for respiratory distress. Clinical scores (p<0.01) and weight gain (p = 0.08) seemed better in the ibuprofen group. Ibuprofen decreased cyclooxygenase, lipoxygenase, and cytochrome P450 products, and increased monoacylglycerols in lung lymph nodes. Ibuprofen modulated the immune response as measured by narrowed range of observed Il-13, Il-17 and IFN-γ gene expression in mediastinal lymph nodes. Lung histology was not different between groups, and viral shedding was increased in calves randomized to ibuprofen. CONCLUSIONS: Ibuprofen decreased PGE2, modulated the immune response, and improved clinical outcomes. However lung histopathology was not affected and viral shedding was increased.

A rapid screen for four corticosteroids in equine synovial fluid.

Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-articularly administered corticosteroids--betamethasone, methylprednisolone, methylprednisolone acetate and triamcinolone acetonide. Sample preparation consisted of protein precipitation followed by a basified liquid-liquid extraction. LC-MS-MS experiments consisted of a six-min isocratic separation using a Phenomenex Polar-RP stationary phase and a mobile phase consisting of 35% acetonitrile, 5 mM ammonium acetate and 0.1% formic acid in nanopure water. The detection system used was a triple quadrupole mass analyzer with thermospray ionization, and compounds were identified using selective reaction monitoring. The method was validated to the ISO/IEC 17025 standard, and real synovial fluid samples were analyzed to demonstrate the application of the method in an antidoping context. The method was highly selective for the four corticosteroids with limits of detection of 1-3 ng/mL. The extraction efficiency was 50-101%, and the matrix effects were 14-31%. These results indicate that the method is a rapid and sensitive screen for the four corticosteroids in equine synovial fluid, fit for purpose for equine antidoping assays.

Identification of protoxins and a microbial basis for red maple (Acer rubrum) toxicosis in equines.

The leaves of Acer rubrum (red maple), especially when wilted in the fall, cause severe oxidative damage to equine erythrocytes, leading to potentially fatal methemoglobinemia and hemolytic anemia. Gallic acid and tannins from A. rubrum leaves have been implicated as the toxic compounds responsible for red maple toxicosis, but the mechanism of action and toxic principle(s) have not been elucidated to date. In order to investigate further how red maple toxicosis occurs, aqueous solutions of gallic acid, tannic acid, and ground dried A. rubrum leaves were incubated with contents of equine ileum, jejunum, cecum, colon, and liver, and then analyzed for the metabolite pyrogallol, as pyrogallol is a more potent oxidizing agent. Gallic acid was observed to be metabolized to pyrogallol maximally in equine ileum contents in the first 24 hr. Incubation of tannic acid and A. rubrum leaves, individually with ileum contents, produced gallic acid and, subsequently, pyrogallol. Ileum suspensions, when passed through a filter to exclude microbes but not enzymes, formed no pyrogallol, suggesting a microbial basis to the pathway. Bacteria isolated from ileum capable of pyrogallol formation were identified as Klebsiella pneumoniae and Enterobacter cloacae. Therefore, gallotannins and free gallic acid are present in A. rubrum leaves and can be metabolized by K. pneumoniae and E. cloacae found in the equine ileum to form pyrogallol either directly or through a gallic acid intermediate (gallotannins). Identification of these compounds and their physiological effects is necessary for the development of effective treatments for red maple toxicosis in equines.