Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.

A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella.

Pathogenic bacteria proliferating inside mammalian host cells need to rapidly adapt to the intracellular environment. How they achieve this and scavenge essential nutrients from the host has been an open question due to the difficulties in distinguishing between bacterial and host metabolites in situ. Here, we capitalized on the inability of mammalian cells to metabolize mannitol to develop a stable isotopic labeling approach to track Salmonella enterica metabolites during intracellular proliferation in host macrophage and epithelial cells. By measuring label incorporation into Salmonella metabolites with liquid chromatography-mass spectrometry (LC-MS), and combining it with metabolic modeling, we identify relevant carbon sources used by Salmonella, uncover routes of their metabolization, and quantify relative reaction rates in central carbon metabolism. Our results underline the importance of the Entner-Doudoroff pathway (EDP) and the phosphoenolpyruvate carboxylase for intracellularly proliferating Salmonella. More broadly, our metabolic labeling strategy opens novel avenues for understanding the metabolism of pathogens inside host cells.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate anatomy, molecular physiology, and control of these movements. We find them driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent system, controlled by an Akt/NO/PKG/A pathway. A concomitant increase in reactive oxygen species and secretion of proteinases and cytokines indicate an inflammation-like state reminiscent of vascular endothelial cells experiencing oscillatory shear stress. This suggests an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals.

Platelet miRNAs: differential expression in coronary artery disease and associations with course of left ventricular systolic function.

BACKGROUND: MicroRNAs are paramount in post transcriptional gene regulation. We investigated platelet miRNAs in patients with CAD and examined potential associations with course of left ventricular ejection fraction (LVEF%). MATERIALS AND METHODS: In a first cohort, 62 MiRNAs were measured in platelets of 100 patients suffering from CAD. Expression profiles of individuals with chronic coronary syndrome (CCS) and MI were compared (CCS n = 67, MI n = 33). Also, associations between miRNA profiles and change in left ventricular ejection fraction (LVEF%) were investigated. In a second cohort of patients suffering from CCS (n = 10), MI (n = 11) or no CAD (n = 13), we measured miRNA expression in platelets, platelet supernatant and serum. This was carried out before and after in vitro platelet activation with CRP. RESULTS: Platelet miRNAs 103a-3p and 155-5p demonstrated higher expression in patients with CCS then in individuals with MI. Furthermore, multiple miRNAs were significantly higher expressed in matched controls compared to MI patients. 8 miRNAs showed higher expression in patients with improving LVEF% after a 1-year follow-up. In our second cohort, we found higher concentrations of 6 miRNAs in the platelet supernatant of patients with CCS, MI and no CAD after in vitro platelet activation. Most of these miRNAs showed a higher abundance in serum of MI patients as compared to CCS. CONCLUSION: Several miRNAs show higher expression in platelets of CCS compared to MI. After in vitro platelet activation, a release of multiple miRNAs out of the thrombocyte was observed. Furthermore, upregulation of serum miRNAs was found in MI patients when compared to CCS patients and individuals without CAD. Hence, platelets could present a source of upregulated circulating miRNAs in MI and additionally affect course of LVEF%.

Principles and Applications of CF2X Moieties as Unconventional Halogen Bond Donors in Medicinal Chemistry, Chemical Biology, and Drug Discovery.

As an orthogonal principle to the established (hetero)aryl halides, we herein highlight the usefulness of CF2X (X = Cl, Br, or I) moieties. Using tool compounds bearing CF2X moieties, we study their chemical/metabolic stability and their logP/solubility, as well as the role of XB in their small molecular crystal structures. Employing QM techniques, we analyze the observed interactions, provide insights into the conformational flexibilities and preferences in the potential interaction space. For their application in molecular design, we characterize their XB donor capacities and its interaction strength dependent on geometric parameters. Implementation of CF2X acetamides into our HEFLibs and biophysical evaluation (STD-NMR/ITC), followed by X-ray analysis, reveals a highly interesting binding mode for fragment 23 in JNK3, featuring an XB of CF2Br toward the P-loop, as well as chalcogen bonds. We suggest that underexplored chemical space combined with unconventional binding modes provides excellent opportunities for patentable chemotypes for therapeutic intervention.

Lower affective empathy in oral contraceptive users: a cross-sectional fMRI study.

Evidence accumulates that oral contraceptive (OC) use modulates various socio-affective behaviors, including empathic abilities. Endogenous and synthetic sex hormones, such as estrogens and progestogens, bind to receptor sites in brain regions (i.e. frontal, limbic, and cerebellar) involved in socio-affective processing. Therefore, the aim of this study was to investigate the role of OC use in empathy. In a cross-sectional functional magnetic resonance imaging study, women in different hormonal states, including OC use (n = 46) or being naturally cycling in the early follicular (fNC: n = 37) or peri-ovulatory phase (oNC: n = 28), performed a visual, sentence-based empathy task. Behaviorally, OC users had lower empathy ratings than oNC women. Congruently, whole-brain analysis revealed significantly larger task-related activation of several brain regions, including the left dorsomedial prefrontal gyrus (dmPFG), left precentral gyrus, and left temporoparietal junction in oNC compared to OC women. In OC users, the activity of the left dmPFG and precentral gyrus was negatively associated with behavioral and self-reported affective empathy. Furthermore, empathy-related region-of-interest analysis indicated negative associations of brain activation with synthetic hormone levels in OC women. Overall, this multimodal, cross-sectional investigation of empathy suggests a role of OC intake in especially affective empathy and highlights the importance of including synthetic hormone levels in OC-related analyses.

Fast accurate quantification of salivary cortisol and cortisone in a large-scale clinical stress study by micro-UHPLC-ESI-MS/MS using a surrogate calibrant approach.

Cortisol and cortisone are common markers for stress and thus preferentially analyzed in matrices that allow non-invasive sampling such as saliva. Though the major drawback of immunoassays is lack of specificity due to cross reactivities, they are still most commonly used for quantification of steroid hormones. To overcome such problems, sensitive methods based on liquid chromatography-mass spectrometry are becoming more and more accepted as the golden standard for steroid bioanalysis as they achieve accurate quantification at trace levels for multiple analytes in the same run. Along this line, the aim of this study was the development of a new microflow UHPLC-ESI-MS/MS method for the measurement of salivary cortisol and cortisone, which due to its microflow regime provides enhanced sensitivity and is more ecofriendly. The developed method implemented sample preparation by Solid-Phase Extraction (SPE) in a 96-well plate format. Data acquisitions were carried out in MRM (multiple reaction monitoring) mode. The quantitative determination of endogenous compounds in saliva remains a challenge since analyte-free matrix is lacking. Hence, a surrogate calibrant approach with cortisol-d4 andcortisone-13C3 was applied for the target compounds in the presented method. A number of factors were optimized and the method validated. The lower limit of quantitation (LLOQ) was 72 and 62 pg mL-1for cortisol and cortisone, respectively. Linear calibration was achieved in the range from 0.062 to 75.5 ng mL-1for cortisol-d4 and 0.072 to 44 ng mL-1forcortisone-13C3. The performance of the method was also evaluated via proficiency test for salivary cortisol. Finally, it was applied successfully to evaluate cortisol and cortisone concentrations in multiple batches in routine clinical stress study samples (4056 total injections with 1983 study samples). Moreover, the instrument performance (in particular retention time variability) within each batch, between different batches and lot-to-lot of 5 investigated capillary columns over time is described. The work documents that micro-UHPLC-ESI-MS/MS is suitable and robust enough to carry out a full clinical study with greater than 1000s of samples over an extended period if adequate internal standards can be used.

No Evidence for a Role of Oral Contraceptive-Use in Emotion Recognition But Higher Negativity Bias in Early Follicular Women.

Accuracy in facial emotion recognition has shown to vary with ovarian hormones, both in naturally cycling women, as well as in women taking oral contraceptives. It remains uncertain however, if specific - endogenous and exogenous - hormonal levels selectively impact recognition of certain basic emotions (or neutral faces) and if this relationship coincides with certain affective states. Therefore, we investigated 86 women under different hormonal conditions and compared their performance in an emotion recognition task as well as self-reported measures of affective states. Based on self-reported cycle days and ovulation testing, the participants have been split into groups of naturally cycling women during their early follicular phase (fNC, n = 30), naturally cycling women during their peri-ovulatory phase (oNC, n = 26), and women taking oral contraceptives (OC, n = 30). Participants were matched for age and did not differ in education or neuropsychological abilities. Self-reported anxiety and depressive affective state scores were similar across groups, but current affective state turned out to be significantly more negative in fNC women. Independent of negative affective state, fNC women showed a significantly higher negativity bias in recognizing neutral faces, resulting in a lower recognition accuracy of neutral faces compared to oNC and OC women. In the OC group only, negative affective state was associated with lower recognition accuracy and longer response times for neutral faces. Furthermore, there was a significant, positive association between disgust recognition accuracy and negative affective state in the fNC group. Low progesterone levels during the early follicular phase were linked to higher negative affective state, whereas in the peri-ovulatory phase they were linked to elevated positive affective state. Overall, previous findings regarding impaired emotion recognition during OC-use were not confirmed. Synthetic hormones did not show a correlation with emotion recognition performance and affective state. Considering the important role of emotion recognition in social communication, the elevated negativity bias in neutral face recognition found for fNC women may adversely impact social interactions in this hormonal phase.

Lipidomic profiling of non-mineralized dental plaque and biofilm by untargeted UHPLC-QTOF-MS/MS and SWATH acquisition.

Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract.

Comprehensive lipidomics of mouse plasma using class-specific surrogate calibrants and SWATH acquisition for large-scale lipid quantification in untargeted analysis.

Lipidomics has gained rising attention in recent years. Several strategies for lipidomic profiling have been developed, with targeted analysis of selected lipid species, typically utilized for lipid quantification by low-resolution triple quadrupole MS/MS, and untargeted analysis by high-resolution MS instruments, focusing on hypothesis generation for prognostic, diagnostic and/or disease-relevant biomarker discovery. The latter methodologies generally yield relative quantification data with limited inter-assay comparability. In this work we aimed to combine untargeted analysis and absolute quantification to enhance data quality and to obtain independent results for optimum comparability to previous studies or database entries. For the lipidomic analysis of mouse plasma, RP-UHPLC hyphenated to a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode was employed. This way, quantifiable data on the MS and the MS/MS level were recorded, which increases assay specificity and quantitative performance. Due to the lack of an appropriate blank matrix for untargeted lipidomics, we herein established a sophisticated strategy for lipid class-specific calibration with stable isotope labeled standards (surrogate calibrants). LLOQs were in the range between 10 and 50 ng mL-1 for LPC, LPE, PI, PS, PG, SM, PC, PE, DAG) or 100-700 ng mL-1 (MAG, TAG), except for cholesterol and CE (1-20 µg mL-1). Acceptable values for accuracy and precision well below ±15% bias were reached for the majority of surrogate calibrants. However, to achieve sufficient accuracy for target lipids, response factors to corresponding surrogate calibrants are required. An approach to estimate response factors via a standard reference material (NIST SRM 1950) was therefore conducted. Furthermore, a useful workflow for post-acquisition re-calibration, involving response factor determination and iteratively built libraries, is suggested. In comparison to single-point calibration, the presented surrogate calibrant method was shown to yield results with improved accuracy that are largely in accordance with standard addition. Quantitative results of real samples (high-fat diet vs control diet) were then compared to two previously published dietary mouse plasma studies that provided absolute lipid levels and showed similar trends.

Guidelines for Selection of Internal Standard-Based Normalization Strategies in Untargeted Lipidomic Profiling by LC-HR-MS/MS.

Due to variation in instrument response caused by various sources of errors throughout an analytical assay, data normalization plays an indispensable role in untargeted LC-MS profiling, yet limited accepted guidelines on this topic exist. In this work, a systematic comparison of several normalization techniques, mainly focusing on internal standard-based approaches, has been performed to derive some general recommendations. For generation of untargeted lipidomic data, a comprehensive ultra-high performance liquid chromatography (UHPLC)-electrospray ionization (ESI)-quadrupole time of flight (QTOF)-MS/MS method was utilized. To monitor instrument stability and evaluate normalization performance, quality control (QC) samples, prepared from aliquots of all experimental samples, were embedded in the sequence. Stable isotope labeled standards, representing differing lipid classes, were spiked to each sample as internal standards for postacquisition normalization. Various metrics were used to compare distinct normalization strategies, with reduction of variation in QC samples being the critical requirement for acceptance of successful normalization. The comparison of intragroup coefficients of variation (CVs), median absolute deviations (MADs), and variance enables simple selection of the best performance of normalization with improved and coherent results. Furthermore, the importance for normalization in critical data sets, showing only minor effects between groups with high variation and outliers, is pointed out. Apart from normalization, also, influences of used raw data types are demonstrated. In addition, effects of various factors throughout the processing workflow were investigated and optimized. Eventually, implementation of quality control samples, even if not required for normalization, provided a useful basis for assessing data quality. Due to lack of consensus for selecting optimum normalization, suggestions for validating data integrity are given.

Comparison of simple monophasic versus classical biphasic extraction protocols for comprehensive UHPLC-MS/MS lipidomic analysis of Hela cells.

In this study, two monophasic isopropanol-water mixtures (IPA:H2O 75:25 v/v and IPA:H2O 90:10 v/v) were compared with traditionally employed biphasic methods of Bligh & Dyer and Matyash et al. as extraction systems for lipidomics analysis in Hela cells. Samples were analyzed by UHPLC-ESI-QTOF-MS/MS in positive and negative mode using sequential window acquisition of all theoretical fragment ion spectra (SWATH) and a relatively new software (MS-DIAL) was employed for the processing of the data which includes detection of peaks, MS/MS spectra deconvolution, identification of detected lipids and alignment of peaks through the analyzed samples. The studied performance parameters such as precision, recoveries of isotopically labeled internal standards and endogenous lipids, number of extracted lipids, and complexity of employed procedure showed that extraction with IPA:H2O 90:10 v/v performs similar to the Matyash protocol and better than Bligh & Dyer as well as IPA:H2O 75:25 v/v. However, less complex monophasic protocol which is simpler to implement and can be executed in plastic rather than glass, make the monophasic IPA:H2O 90:10 v/v protocol an excellent alternative to the classical biphasic protocols for reversed phase LC-MS lipidomics studies.

Quantification of steroid hormones in plasma using a surrogate calibrant approach and UHPLC-ESI-QTOF-MS/MS with SWATH-acquisition combined with untargeted profiling.

In spite of demonstrated lack of accuracy and consistency, quantification of steroid hormones is still most commonly executed via immunoassays. Mass spectrometric methods with triple quadrupole instruments are well established and, because of their proven robustness and sensitivity, best suited for targeted analysis. However, recent studies have shown that high-resolution mass spectrometers, like quadrupole time-of-flight instruments (QTOF), show comparable performance in terms of quantification and can generate additional sample information via untargeted profiling workflows. We demonstrate that adequate accuracy and selectivity for estradiol and testosterone can be achieved with a QTOF by data-independent acquisition with sequential window acquisition of all theoretical fragment-ion mass spectra (SWATH). Besides potential combination of targeted quantification and untargeted profiling, SWATH offers advantages with respect to sensitivity because the reduced total number of MS/MS experiments could be used to increase accumulation time without increasing cycle time. By applying a surrogate calibrant method leading to successful validation, a reliable method for absolute steroid quantification and high potential for steroid profiling has been developed. Linear calibration was achieved in the range from 10 to 1,000 pg mL-1 for 13C3-estradiol and from 20 to 15,000 pg mL-1 for 13C3-testosterone. Results for inter-day precision (13C3-estradiol: 4.5-10.2%; 13C3-testosterone: 5.1-7.8%) and inter-day accuracy (13C3-estradiol: 94.6-112.8%; 13C3-testosterone: 98.2-107.7%) were found to be well acceptable. Eventually, the method has been utilized to measure clinical samples of a study in which male volunteers obtained transdermal estradiol patches and sex hormone levels were quantified in plasma.

Insulin and Estrogen Independently and Differentially Reduce Macronutrient Intake in Healthy Men.

Context: Insulin administration to the central nervous system inhibits food intake, but this effect has been found to be less pronounced in female compared with male organisms. This sex-specific pattern has been suggested to arise from a modulating influence of estrogen signaling on the insulin effect. Objective: We assessed in healthy young men whether pretreatment with transdermal estradiol interacts with the hypophagic effect of central nervous insulin administration via the intranasal pathway. Design, Setting, Participants, and Intervention: According to a 2×2 design, two groups of men (n = 16 in each group) received a 3-day transdermal estradiol (100 µg/24 h) or placebo pretreatment and on two separate mornings were intranasally administered 160 IU regular human insulin or placebo. Main Outcome Measures: We assessed free-choice ad libitum calorie intake from a rich breakfast buffet and relevant blood parameters in samples collected before and after breakfast. Results: Estrogen treatment induced a 3.5-fold increase in serum estradiol concentrations and suppressed serum testosterone concentrations by 70%. Independent of estradiol administration, intranasal insulin reduced the intake of carbohydrates during breakfast, attenuating in particular the consumption of sweet, palatable foods. Estradiol treatment per se decreased protein consumption. We did not find indicators of eating-related interactions between both hormones. Conclusions: Results indicate that, in an acute setting, estrogen does not interact with central nervous insulin signaling in the control of eating behavior in healthy men. Insulin and estradiol rather exert independent inhibiting effects on macronutrient intake.