Protocol for developing a core outcome set for male infertility research: an international consensus development study.

STUDY QUESTION: We aim to develop, disseminate and implement a minimum data set, known as a core outcome set, for future male infertility research. WHAT IS KNOWN ALREADY: Research into male infertility can be challenging to design, conduct and report. Evidence from randomized trials can be difficult to interpret and of limited ability to inform clinical practice for numerous reasons. These may include complex issues, such as variation in outcome measures and outcome reporting bias, as well as failure to consider the perspectives of men and their partners with lived experience of fertility problems. Previously, the Core Outcome Measure for Infertility Trials (COMMIT) initiative, an international consortium of researchers, healthcare professionals and people with fertility problems, has developed a core outcome set for general infertility research. Now, a bespoke core outcome set for male infertility is required to address the unique challenges pertinent to male infertility research. STUDY DESIGN SIZE DURATION: Stakeholders, including healthcare professionals, allied healthcare professionals, scientists, researchers and people with fertility problems, will be invited to participate. Formal consensus science methods will be used, including the modified Delphi method, modified Nominal Group Technique and the National Institutes of Health's consensus development conference. PARTICIPANTS/MATERIALS SETTING METHODS: An international steering group, including the relevant stakeholders outlined above, has been established to guide the development of this core outcome set. Possible core outcomes will be identified by undertaking a systematic review of randomized controlled trials evaluating potential treatments for male factor infertility. These outcomes will be entered into a modified Delphi method. Repeated reflection and re-scoring should promote convergence towards consensus outcomes, which will be prioritized during a consensus development meeting to identify a final core outcome set. We will establish standardized definitions and recommend high-quality measurement instruments for individual core outcomes. STUDY FUNDING/COMPETING INTERESTS: This work has been supported by the Urology Foundation small project award, 2021. C.L.R.B. is the recipient of a BMGF grant and received consultancy fees from Exscentia and Exceed sperm testing, paid to the University of Dundee and speaking fees or honoraria paid personally by Ferring, Copper Surgical and RBMO. S.B. received royalties from Cambridge University Press, Speaker honoraria for Obstetrical and Gynaecological Society of Singapore, Merk SMART Masterclass and Merk FERRING Forum, paid to the University of Aberdeen. Payment for leadership roles within NHS Grampian, previously paid to self, now paid to University of Aberdeen. An Honorarium is received as Editor in Chief of Human Reproduction Open. M.L.E. is an advisor to the companies Hannah and Ro. B.W.M. received an investigator grant from the NHMRC, No: GNT1176437 is a paid consultant for ObsEva and has received research funding from Ferring and Merck. R.R.H. received royalties from Elsevier for a book, consultancy fees from Glyciome, and presentation fees from GryNumber Health and Aytu Bioscience. Aytu Bioscience also funded MiOXYS systems and sensors. Attendance at Fertility 2020 and Roadshow South Africa by Ralf Henkel was funded by LogixX Pharma Ltd. R.R.H. is also Editor in Chief of Andrologia and has been an employee of LogixX Pharma Ltd. since 2020. M.S.K. is an associate editor with Human Reproduction Open. K.Mc.E. received an honoraria for lectures from Bayer and Pharmasure in 2019 and payment for an ESHRE grant review in 2019. His attendance at ESHRE 2019 and AUA 2019 was sponsored by Pharmasure and Bayer, respectively. The remaining authors declare no competing interests. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials (COMET) initiative registration No: 1586. Available at www.comet-initiative.org/Studies/Details/1586. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

Immobilization of natural lipid biomembranes and their interactions with choline carboxylates. A nanoplasmonic sensing study.

The cell membrane is mainly composed of lipid bilayers with inserted proteins and carbohydrates. Lipid bilayers made of purified or synthetic lipids are widely used for estimating the effect of target compounds on cell membranes. However, the composition of such biomimetic membranes is much simpler than the composition of biological membranes. Interactions between compounds and simple composition biomimetic membranes might not demonstrate the effect of target compounds as precisely as membranes with compositions close to real organisms. Therefore, the aim of our study is to construct biomimetic membrane closely mimicking the state of natural membranes. Liposomes were prepared from lipids extracted from L-α-phosphatidylcholine, Escherichia coli, yeast (Saccharomyces cerevisiae) and bovine liver cells through agitation and sonication. They were immobilized onto silicon dioxide (SiO2) sensor surfaces using N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer with calcium chloride. The biomimetic membranes were successfully immobilized onto the SiO2 sensor surface and detected by nanoplasmonic sensing. The immobilized membranes were exposed to choline carboxylates. The membrane disruption effect was, as expected, more pronounced with increasing carbohydrate chain length of the carboxylates. The results correlated with the toxicity values determined using Vibrio fischeri bacteria. The yeast extracted lipid membranes had the strongest response to introduction of choline laurate while the bovine liver lipid extracted liposomes were the most sensitive towards the shorter choline carboxylates. This implies that the composition of the cell membrane plays a crucial role upon interaction with choline carboxylates, and underlines the necessity of testing membrane systems of different origin to obtain an overall image of such interactions.

Interactions of Ionic Liquids and Spirocyclic Compounds with Liposome Model Membranes. A Steady-State Fluorescence Anisotropy Study.

Understanding the toxicity of ionic liquids (ILs) is crucial in the search of greener chemicals. By comparing in vivo toxicity and in vitro interactions determined between compounds and biomimetic lipid membranes, more detailed toxicity vs. structure relation can be obtained. However, determining the interactions between non-surface-active compounds and liposomes has been a challenging task. Organisational changes induced by ILs and IL-like spirocyclic compounds within 1,6-diphenyl-1,3,5-hexatriene-doped biomimetic liposomes was studied by steady-state fluorescence anisotropy technique. The extent of organisational changes detected within the liposome bilayers were compared to the toxicity of the compounds determined using Vibrio Fischeri bacteria. Four liposome compositions made of pure 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocoline (POPC) and mixtures of POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and cholesterol (Chol) were tested as biomimetic models. Changes observed within the POPC/POPS/Chol 55:20:25 bilayers correlated the best with the toxicity results: ten out of twelve compounds followed the trend of increasing bilayer disorder - increasing toxicity. The study suggests that the toxicity of non-surface-active compounds is dependent on their ability to diffuse into the bilayers. The extent of bilayer's organisational changes correlates rather well with the toxicity of the compounds. Highly sensitive technique, such as fluorescence anisotropy measurements, is needed for detecting subtle changes within the bilayer structures.

Correlation between Ionic Liquid Cytotoxicity and Liposome-Ionic Liquid Interactions.

This study aims at extending the understanding of the toxicity mechanism of ionic liquids (ILs) using various analytical methods and cytotoxicity assays. The cytotoxicity of eight ILs and one zwitterionic compound was determined using mammalian and bacterial cells. The time dependency of the IL toxicity was assessed using human corneal epithelial cells. Hemolysis was performed using human red blood cells and the results were compared with destabilization data of synthetic liposomes upon addition of ILs. The effect of the ILs on the size and zeta potential of liposomes revealed information on changes in the lipid bilayer. Differential scanning calorimetry was used to study the penetration of the ILs into the lipid bilayer. Pulsed field gradient nuclear magnetic resonance spectroscopy was used to determine whether the ILs occurred as unimers, micelles, or if they were bound to liposomes. The results show that the investigated ILs can be divided into three groups based on the cytotoxicity mechanism: cell wall disrupting ILs, ILs exerting toxicity through both cell wall penetration and metabolic alteration, and ILs affecting solely on cell metabolism.

WtF-Nano: One-Pot Dewatering and Water-Free Topochemical Modification of Nanocellulose in Ionic Liquids or γ-Valerolactone.

Ionic liquids are used to dewater a suspension of birch Kraft pulp cellulose nanofibrils (CNF) and as a medium for water-free topochemical modification of the nanocellulose (a process denoted as "WtF-Nano"). Acetylation was applied as a model reaction to investigate the degree of modification and scope of effective ionic liquid structures. Little difference in reactivity was observed when water was removed, after introduction of an ionic liquid or molecular co-solvent. However, the viscoelastic properties of the CNF suspended in two ionic liquids show that the more basic, but non-dissolving ionic liquid, allows for better solvation of the CNF. Vibrio fischeri bacterial tests show that all ionic liquids in this study were harmless. Scanning electron microscopy and wide-angle X-ray scattering on regenerated samples show that the acetylated CNF is still in a fibrillar form. 1 D and 2 D NMR analyses, after direct dissolution in a novel ionic liquid electrolyte solution, indicate that both cellulose and residual xylan on the surface of the nanofibrils reacts to give acetate esters.

Cholesterol affects the interaction between an ionic liquid and phospholipid vesicles. A study by differential scanning calorimetry and nanoplasmonic sensing.

The present work aims at studying the interactions between cholesterol-rich phosphatidylcholine-based lipid vesicles and trioctylmethylphosphonium acetate ([P8881][OAc]), a biomass dissolving ionic liquid (IL). The effect of cholesterol was assayed by using differential scanning calorimetry (DSC) and nanoplasmonic sensing (NPS) measurement techniques. Cholesterol-enriched dipalmitoyl-phosphatidylcholine vesicles were exposed to different concentrations of the IL, and the derived membrane perturbation was monitored by DSC. The calorimetric data could suggest that the binding and infiltration of the IL are delayed in the vesicles containing cholesterol. To clarify our findings, NPS was applied to quantitatively follow the resistance of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine incorporating 0, 10, and 50mol% of cholesterol toward the IL exposure over time. The membrane perturbation induced by different concentrations of IL was found to be a concentration dependent process on cholesterol-free lipid vesicles. Moreover, our results showed that lipid depletion in cholesterol-enriched lipid vesicles is inversely proportional to the increasing amount of cholesterol in the vesicles. These findings support that cholesterol-rich lipid bilayers are less susceptible toward membrane disrupting agents as compared to membranes that do not incorporate any sterols. This probably occurs because cholesterol tightens the phospholipid acyl chain packing of the plasma membranes, increasing their resistance and reducing their permeability.

Pure Glaucoma Drugs Are Toxic to Immortalized Human Corneal Epithelial Cells, but They Do Not Destabilize Lipid Membranes.

PURPOSE: Most pure glaucoma drugs (pGDs) are hydrophobic substances intended to reduce elevated intraocular pressure. The aims of our study were to determine the toxicity of pGDs (brimonidine tartrate, brinzolamide, latanoprost, timolol maleate, and pilocarpine hydrochloride) on ocular surface cells and to establish whether their toxicity is subsequent to cellular membrane destabilization. METHODS: The toxicity of clinically efficient doses of pGDs was measured at different time points in a cell culture of human corneal epithelial cells using a redox indicator. pGD interaction with the plasma membrane was analyzed using a hemolysis assay and liposome electrokinetic chromatography. The capacity of pGDs to induce endoplasmic reticulum stress was investigated by immunoblotting. RESULTS: The toxicity assay showed that all pGDs decrease the viability of the epithelial cells to variable degrees. Early toxicity was measured for 4% pilocarpine and 0.15% brimonidine with 60% cell death at 4 hours, whereas 2% pilocarpine and 0.005% latanoprost showed almost 100% toxicity but only after 16 hours. The hemolysis assay and liposome electrokinetic chromatography experiments suggested that interaction between pGDs and lipid membranes is weak and cannot explain cell death through lysis. Immunoblotting revealed that the drugs activate endoplasmic reticulum stress and, with the exception of pilocarpine, have the capacity to induce apoptosis through upregulation of C/EBP homologous protein. CONCLUSIONS: Our study indicates that all studied pGDs decrease the viability of the corneal epithelial cells, but none of the tested compounds were able to destabilize cellular membranes. The pGDs seem to be internalized and can induce apoptosis through C/EBP homologous protein recruitment.

Impact of Surface-Active Guanidinium-, Tetramethylguanidinium-, and Cholinium-Based Ionic Liquids on Vibrio Fischeri Cells and Dipalmitoylphosphatidylcholine Liposomes.

We investigated the toxicological effect of seven novel cholinium, guanidinium, and tetramethylguanidinium carboxylate ionic liquids (ILs) from an ecotoxicological point of view. The emphasis was on the potential structure-toxicity dependency of these surface-active ILs in aqueous environment. The median effective concentrations (EC50) were defined for each IL using Vibrio (Aliivibrio) fischeri marine bacteria. Dipalmitoylphosphatidylcholine (DPPC) liposomes were used as biomimetic lipid membranes to study the interactions between the surface-active ILs and the liposomes. The interactions were investigated by following the change in the DPPC phase transition behaviour using differential scanning calorimetry (DSC). Critical micelle concentrations for the ILs were determined to clarify the analysis of the toxicity and the interaction results. Increasing anion alkyl chain length increased the toxicity, whereas branching of the chain decreased the toxicity of the ILs. The toxicity of the ILs in this study was mainly determined by the surface-active anions, while cations induced a minor impact on the toxicity. In the DSC experiments the same trend was observed for all the studied anions, whereas the cations seemed to induce more variable impact on the phase transition behaviour. Toxicity measurements combined with liposome interaction studies can provide a valuable tool for assessing the mechanism of toxicity.

The Effect of Phospholipids on Tear Film Lipid Layer Surface Activity.

Purpose: We illustrate the importance of small quantities (<10 mol%) of polar phospholipids on the surface-active behavior of meibum-like lipid compositions. Methods: Artificial meibum-like lipid mixture containing cholesteryl and wax esters was mixed with differing amounts of phosphatidylcholine (PC). The surface activity of these mixtures was investigated at the air-water interface by recording surface pressure created by the lipid layer as a function of molecular area at 37°C. The PC proportion in the mixtures was 0, 2.5, 5, 7.5, or 10 mol%, and the remaining proportion in the mixture was 50:50 (mol/mol) of cholesteryl oleate (CO) and behenyl oleate (BO). Also, the effect of temperature was investigated. Results: The surface activity of the mixtures increased in a very predictable and consistent fashion as a function of the PC proportion. The lipid mixture containing only CO and BO showed miniscule surface activity. However, already 2.5% PC mixed with the nonpolar CO and BO generated considerable increase in surface pressure. At small surface areas, the behavior of 7.5% and 10% PC compositions started to approach that of a pure PC monolayer. The temperature did not have a considerable impact on the surface-active behavior of the PC-containing compositions. Conclusions: The polar phospholipids have a considerable effect on the surface-active properties of artificial tear film lipid layer (TFLL) compositions. Surprisingly, this takes place already at very low and physiologically relevant PC proportions. The effect is more dependent on the actual amount of the phospholipids at the air-tear interface than on the relative amount of these lipids in TFLL.

Distribution of local anesthetics between aqueous and liposome phases.

Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (KD) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25°C, the distribution constants were determined at 37 and 42°C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant.

Monoliths in capillary electrochromatography and capillary liquid chromatography in conjunction with mass spectrometry.

Here, we have reviewed separation studies utilizing monolithic capillary columns for separation of compounds preceding MS analysis. The review is divided in two parts according to the used separation method, namely CEC and capillary LC (cLC). Based on our overview, monolithic CEC-MS technique have been more focused on the syntheses of highly specialized and selective separation phase materials for fast and efficient separation of specific types of analytes. In contrast, monolithic cLC-MS is more widely used and is often employed, for instance, in the analysis of oligonucleotides, metabolites, and peptides and proteins in proteomic studies. While poly(styrene-divinylbenzene)-based and silica-based monolithic capillaries found their place in proteomic analyses, the other laboratory-synthesized monoliths still wait for their wider utilization in routine analyses. The development of new monolithic materials will most likely continue due to the demand of more efficient and rapid separation of increasingly complex samples.

Systemic differences in serum metabolome: a cross sectional comparison of women with localised and widespread pain and controls.

Chronic musculoskeletal pain exists either as localised to a single region or as widespread to multiple sites in several quadrants of the body. Prospective studies indicate that widespread pain could act as a far end of a continuum of musculoskeletal pain that started with chronic localised pain. The mechanism by which the transition from localised pain to widespread occurs is not clear, although many studies suggest it to be an altered metabolism. In this study, systemic metabolic differences between women with chronic localised neck-shoulder pain (NP), women with chronic widespread pain (CWP) and women who were healthy (CON) were assessed. Blood samples were analysed taking a metabolomics approach using gas chromatography mass spectrometry (GC-MS) and orthogonal partial least square discriminant analysis (OPLS-DA). The metabolomics analysis showed a clear systematic difference in the metabolic profiles between the subjects with NP and the CON but only a weak systematic difference between the subjects with CWP and the CON. This most likely reflects a difference in the portion of the metabolome influenced by the two pain conditions. In the NP group, the overall metabolic profile suggests that processes related to energy utilisation and lipid metabolism could be central aspects of mechanisms maintaining disorder.

The Effect of Ambient Ozone on Unsaturated Tear Film Wax Esters.

PURPOSE: Tear film lipid layer (TFLL) is constantly exposed to reactive ozone in the surrounding air, which may have detrimental effects on ocular health. Behenyl oleate (BO), a representative tear film wax ester, was used to study the reaction with ozone at the air-water interface. METHODS: Time-dependent changes in mean molecular area of BO monolayers were measured at different ozone concentrations and surface pressures. In addition, the effect of ascorbic acid on the reaction rate was determined. Reaction was followed using thin-layer chromatography and reaction products were identified using liquid chromatography-electrospray ionization mass spectrometry (LC-MS). Tear fluid samples from healthy subjects were analyzed with LC-MS for any ozonolysis reaction products. RESULTS: Behenyl oleate was found to undergo rapid ozonolysis at the air-water interface at normal indoor ozone concentrations. The reaction was observed as an initial expansion followed by a contraction of the film area. Ascorbic acid was found to decrease the rate of ozonolysis. Main reaction products were identified as behenyl 9-oxononanoate and behenyl 8-(5-octyl-1,2,4-trioxolan-3-yl)octanoate. Similar ozonolysis products were not detected in the tear fluid samples. CONCLUSIONS: At the air-water interface, unsaturated wax esters react readily with ozone in ambient air. However, no signs of ozonolysis products were found in the tear fluid. This is most likely due to the antioxidant systems present in tear fluid. Last, the results show that ozonolysis needs to be controlled in future surface chemistry studies on tear film lipids.

Surface properties of artificial tear film lipid layers: effects of wax esters.

PURPOSE: The tear film lipid layer is believed to stabilize the tear film and to retard evaporation. Based on previous simple in vitro studies, the evidence for the latter property is scarce. In this study, we used complex lipid mixtures including various wax esters to study their physical properties and evaporation retarding effect. METHODS: Twelve samples of artificial tear film lipid layer mixtures composed of (L-α)-phosphatidylcholine, cholesterol oleate, and triglycerides were mixed with wax esters. A Langmuir balance was used to analyze the compressibility and rheological properties of these mixtures. In addition, a custom-built system was used for the evaporation studies used at 35°C. Lipid films were imaged with Brewster angle microscopy. RESULTS: None of the studied lipid mixtures decreased the evaporation rate. All lipid mixtures had similar compression isotherms and viscoelastic properties regardless of the wax ester species or its concentration. The results suggest that the overall properties of these mixtures are independent of individual lipid species and that these films are very cooperative and showed minor variation depending on the wax ester species. Brewster angle microscopy images revealed that the lipid films assembled into multiple layers. CONCLUSIONS: Wax ester-containing lipid mixtures resembling the tear film lipid layer are organized in a layered fashion so that amphiphilic lipids are adjacent to the aqueous phase and the nonpolar lipids are layered on top of these. This organization does not retard evaporation and raises overall questions about the role of lipids in the tear film.

Antievaporative mechanism of wax esters: implications for the function of tear fluid.

The tear film lipid layer (TFLL) is considered to act as an evaporation barrier and to maintain the tear film intact between blinks. In vitro methods have, however, failed to reproduce this evaporation-retarding effect. Wax esters (WEs) are a major component of the TFLL. Close to their bulk melting temperature, WEs have been found to retard the evaporation of water, but the nature of this mechanism has remained unclear. We studied the interfacial organization of WE films by measuring their isochors and isotherms and evaporation-retarding effect, and we imaged these films by Brewster angle microscopy (BAM). Behenyl palmitoleate (BP) was used as a representative WE because it resembles the WEs found in meibum. At low temperatures, BP forms solid monolayer crystals in which the molecules are organized in a bulk-like extended conformation. Within approximately 3 °C below the bulk melting temperature, these solid monolayer domains coexist with a fluid monolayer film. At temperatures above the bulk melting temperature, BP forms a completely fluid monolayer in which the molecules are in a hairpin conformation. A fluid hairpin monolayer of BP does not significantly retard evaporation, whereas a solid monolayer decreases evaporation by >50%. The results provide a molecular-level rationale for the evaporation-retarding properties of WEs close to their melting temperature.

Lipid-modifying enzymes in human tear fluid and corneal epithelial stress response.

PURPOSE: Since homeostasis at the ocular surface requires a delicate balance between numerous factors, and the external environment contributes as an unpredictable component, we aimed to understand the role that various lipids and their regulators have in the complex process that maintains a healthy corneal surface. METHODS: Through basic proteomics, we tested the presence of sphingolipid metabolism enzymes in normal human tears, and then used a cell culture model to study how the proteins are secreted and for what purpose. RESULTS: When studying healthy tears, we found that sphingolipid-specific enzymes, acid and neutral sphingomyelinases, and ceramidases can be detected. The role played by sphingolipid metabolism in stress provided the motivation for further studies concerning their secretion/leakage in the extracellular environment in a cell culture model of human corneal epithelial cells (HCE). Among the stress agents investigated (i.e., ultraviolet B [UV-B] radiation, hyperosmolarity [HO], and lipopolysaccharide [LPS]), UV-B and HO induced dose-dependent release/secretion of sphingomyelinases from the cells. In an attempt to identify the route of secretion or release of the enzyme, we discovered that the tested stress stimuli induced shedding of extracellular vesicles in the HCE-conditioned medium. CONCLUSIONS: Extracellular stress affects tear fluid composition more profoundly than just secretion of proinflammatory mediators. Lipids at the ocular surface, either in tear fluid or within the corneal epithelial cells, can be modified by a relatively large array of lipases to modulate their functions. Moreover, extracellular vesicles in the tear fluid could represent a valuable noninvasive diagnosis tool for anterior segment diseases.

Comparative metabolomics of muscle interstitium fluid in human trapezius myalgia: an in vivo microdialysis study.

PURPOSE: The mechanisms behind trapezius myalgia are unclear. Many hypotheses have been presented suggesting an altered metabolism in the muscle. Here, muscle microdialysate from healthy and myalgic muscle is analysed using metabolomics. Metabolomics analyse a vast number of metabolites, enabling a comprehensive explorative screening of the cellular processes in the muscle. METHODS: Microdialysate samples were obtained from the shoulder muscle of healthy and myalgic subjects that performed a work and stress test. Samples from the baseline period and from the recovery period were analysed using gas chromatography­mass spectrometry (GC­MS) together with multivariate analysis to detect differences in extracellular content of metabolites between groups. Systematic differences in metabolites between groups were identified using multivariate analysis and orthogonal partial least square discriminate analysis (OPLS-DA). A complementary Mann­Whitney U test of group difference in individual metabolites was also performed. RESULTS: A large number of metabolites were detected and identified in this screening study. At baseline, no systematic differences between groups were observed according to the OPLS-DA. However, two metabolites, l-leucine and pyroglutamic acid, were significantly more abundant in the myalgic muscle compared to the healthy muscle. In the recovery period, systematic difference in metabolites between the groups was observed according to the OPLS-DA. The groups differed in amino acids, fatty acids and carbohydrates. Myristic acid and putrescine were significantly more abundant and beta-d-glucopyranose was significantly less abundant in the myalgic muscle. CONCLUSION: This study provides important information regarding the metabolite content, thereby presenting new clues regarding the pathophysiology of the myalgic muscle.

Melting points--the key to the anti-evaporative effect of the tear film wax esters.

PURPOSE: We examined in vitro the evaporation-retarding effect of wax esters (WEs). The WEs resembled closely the most abundant WE species in meibum. METHODS: A custom-built system was used to measure the evaporation rates through WE layers applied to the air-water interface at 35°C and, as a reference, at 30°C and 41°C. Additionally, the melting points of the WEs were determined. The organization and stability of the WE layers were assessed using Brewster angle microscopy (BAM) and Langmuir film experiments, respectively. RESULTS: Four of 19 WEs retarded evaporation at 35°C: behenyl palmitoleate (BP), behenyl oleate (BO), behenyl linoleate (BLN), and behenyl linolenate (BLNN) decreased evaporation by 20% to 40%. BP was the most effective evaporation retardant. At 30°C the most effective retardants were BLN and BLNN decreasing evaporation by ~50%, whereas BP and BO decreased evaporation by only 5% to 10%. At 41°C, each lipid decreased evaporation by only 2% to 4%. The evaporation-retardant WEs all melted within 2°C of physiological temperature. BAM images showed that the evaporation-retardant WE layers spread somewhat uniformly and possibly exhibited areas of condensed lipid. The isotherms suggested that WE layers were surface pressure tolerant but unstable under compression-relaxation cycles. CONCLUSIONS: The evaporation-retarding effect is dependent on the physicochemical properties of the WEs at given temperature, and therefore, the effect most likely arises from a certain phase of the WE layer. However, WEs as such are poor surfactants and need to be accompanied by polar lipids to form stable lipid layers.

Integrated histopathological and urinary metabonomic investigation of the pathogenesis of microcystin-LR toxicosis.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Do lipids retard the evaporation of the tear fluid?

PURPOSE: We examined in vitro the potential evaporation-retarding effect of the tear film lipid layer (TFLL). The artificial TFLL compositions used here were based on the present knowledge of TFLL composition. METHODS: A custom-built system was developed to measure evaporation rates at 35°C. Lipids were applied to an air-water interface, and the evaporation rate through the lipid layer was defined as water loss from the interface. A thick layer of olive oil and a monolayer of long-chain alcohol were used as controls. The artificial TFLLs were composed of 1 to 4 lipid species: polar phosphatidylcholine (PC), nonpolar cholesteryl ester, triglycerides, and wax ester (WE). Brewster angle microscopy (BAM) and interfacial shear rheometry (ISR) were used to assess the lateral structure and shear stress response of the lipid layers, respectively. RESULTS: Olive oil and long-chain alcohol decreased evaporation by 54% and 45%, respectively. The PC monolayer and the four-component mixtures did not retard evaporation. WE was the most important evaporation-retardant TFLL lipid (∼20% decrease). In PC/WE mixtures, an ∼90% proportion of WE was required for evaporation retardation. Based on BAM and ISR, WE resulted in more condensed layers than the non-retardant layers. CONCLUSIONS: Highly condensed, solid-like lipid layers, such as those containing high proportions of WEs, are evaporation-retardant. In multi-component lipid layers, the evaporation-retardant interactions between carbon chains decrease and, therefore, these lipid layers do not retard evaporation.