UV photo-degradation of the secondary lichen substance parietin: A multi-spectroscopic analysis in astrobiology perspective.

The cortical anthraquinone yellow-orange pigment parietin is a secondary lichen substance providing UV-shielding properties that is produced by several lichen species. In our work, the secondary metabolite has been extracted from air-dried thalli of Xanthoria parietina. The aims of this study were to characterize parietin absorbance through UV-VIS spectrophotometry and with IR spectroscopy and to evaluate its photodegradability under UV radiation through in situ reflectance IR spectroscopy to understand to what extent the substance may have a photoprotective role. This allows us to relate parietin photo-degradability to the lichen UV tolerance in its natural terrestrial habitat and in extreme environments relevant for astrobiology such as Mars. Extracted crystals were UV irradiated for 5.59 h under N2 flux. After the UV irradiation, we assessed relevant degradations in the 1614, 1227, 1202, 1160 and 755 cm-1 bands. However, in light of Xanthoria parietina survivability in extreme conditions such as space- and Mars-simulated ones, we highlight parietin UV photo-resistance and its relevance for astrobiology as photo-protective substance and possible bio-hint.

R2R01: A long-acting single-chain peptide agonist of RXFP1 for renal and cardiovascular diseases.

BACKGROUND: The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half-life of serelaxin. Optimization of fatty acid-acetylated single-chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half-life. EXPERIMENTAL APPROACH: Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo-allergic reactions and CD4+ T-cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs. KEY RESULTS: In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half-life. The in vitro assays with LAD2 cells and CD4+ T-cells showed that R2R01 had low potential for pseudo-allergic and immunogenic reactions, respectively. CONCLUSION AND IMPLICATIONS: R2R01 is a potent RXFP1 agonist with an extended half-life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.

Interleukin 23 receptor: Expression and regulation in immune cells.

The importance of IL-23 and its specific receptor, IL-23R, in the pathogenesis of several chronic inflammatory diseases has been established, but the underlying pathological mechanisms are not fully understood. This review focuses on IL-23R expression and regulation in immune cells.

Resilience of Xanthoria parietina under Mars-like conditions: photosynthesis and oxidative stress response.

MAIN CONCLUSION: Xanthoria parietina survivability in Mars-like conditions was supported by water-lysis efficiency recovery and antioxidant content balancing with ROS production after 30 days of exposure. Xanthoria parietina (L.) Th. Fr. is a widespread lichen showing tolerance against air pollutants and UV-radiation. It has been tested under space-like and Mars-like conditions resulting in high recovery performances. Hereby, we aim to assess the mechanisms at the basis of the thalli resilience against multiple space stress factors. Living thalli of X. parietina were exposed to simulated Martian atmospheric conditions (Dark Mars) and UV radiation (Full Mars). Then, we monitored as vitality indicator the photosynthetic efficiency, assessed by in vivo chlorophyll emission fluorescence measurements (FM; FV/F0). The physiological defense was evaluated by analyzing the thalli antioxidant capacity. The drop of FM and FV/F0 immediately after the exposure indicated a reduction of photosynthesis. After 24 h from exposure, photosynthetic efficiency began to recover suggesting the occurrence of protective mechanisms. Antioxidant concentrations were higher during the exposure, only decreasing after 30 days. The recovery of photosynthetic efficiency in both treatments suggested a strong resilience by the photosynthetic apparatus against combined space stress factors, likely due to the boosted antioxidants at the beginning and their depletion at the end of the exposure. The overall results indicated that the production of antioxidants, along with the occurrence of photoprotection mechanisms, guarantee X. parietina survivability in Mars-like environment.

Mercury accumulation efficiency of different biomonitors in indoor environments: the case study of the Central Italian Herbarium (Florence, Italy).

Biomonitoring studies are often employed to track airborne pollutants both in outdoor and indoor environments. In this study, the mercury (Hg) sorption by three biomonitors, i.e., Pinus nigra bark, Pseudovernia furfuracea lichen, and Hypnum cupressiforme moss, was investigated in controlled (indoor) conditions. In comparison to outdoor environments, controlled conditions offer the opportunity to investigate more in detail the variables (humidity, temperature, pollutants speciation, etc.) that control Hg uptake. The biomonitors were exposed in two distinct periods of the year for 2 and 12 months respectively, in the halls of the Central Italian Herbarium (Natural History Museum of the University of Florence, Italy), which are polluted by Hg, due to past plant sample treatments. The Hg sorption trend was monitored every 3 weeks by recording: (i) the Hg content in the substrata, (ii) gaseous elemental mercury (GEM) concentrations in the exposition halls, (iii) temperature, (iv) humidity, and (v) particulate matter (PM) concentrations. At the end of the experiment, Hg concentrations in the biomonitors range from 1130 ± 201 to 293 ± 45 µg kg-1 (max-min) in barks, from 3470 ± 571 to 648 ± 40 µg kg-1 in lichens, and from 3052 ± 483 to 750 ± 127 µg kg-1 in mosses. All the biomonitors showed the highest Hg accumulation after the first 3 weeks of exposure. Mercury concentrations increased over time showing a continuous accumulation during the experiments. The biomonitors demonstrated different Hg accumulation trends in response to GEM concentrations and to the different climatic conditions (temperature and humidity) of the Herbarium halls. Barks strictly reflected the gaseous Hg pollution, while lichen and moss accumulation was also influenced by the climatic conditions of the indoor environment. Mercury bound to PM seemed to provide a negligible contribution to the biomonitors final uptake.

T cells in the pathogenesis of axial spondyloarthritis.

Axial spondyloarthritis (axSpA) is the prototype of the spondyloarthritis spectrum. The involvement of T cells in its pathogenesis has long been suspected on the basis of the association with the major histocompatibility complex I molecule HLA-B27 and the pivotal role of interleukin 17 in the inflammatory mechanisms associated with the disease. Moreover, the presence of unconventional or "innate-like" T cells within the axial enthesis suggests an important role for these cells in the pathophysiology of the disease. In this review, we describe the characteristics and the interleukin 17 secretion capacity of the T-cell subsets identified in axSpA. We discuss the genetic and epigenetic mechanisms that support the alteration of T-cell functions and promote their activation in axSpA. We also discuss recent data on T cells that could explain the extra-articular manifestations of the SpA spectrum.

Sileneisabellae (Caryophyllaceae), a new campion species from serpentine soils of Albania.

The new species Sileneisabellae is described and illustrated from the Skënderbëut mountain range of central Albania. It grows on the ultramafic mountain slopes around Qafë Shtamë, in the understorey of open Pinusnigra forests and in the rocky grasslands above the forest belt, at 1000-1600 m a.s.l. Sileneisabellae is a serpentine endemic likely belonging to section Elisanthe (Fenzl ex Endl.) Ledeb. and shows affinities with the widespread European species S.noctiflora L. It is sharply distinct from the latter species in habit, stem and leaf pubescence, morphology, and biology of the flowers and length of the carpophore. Moreover, the ecology of the two taxa is also contrasting, being S.noctiflora a synanthropic-ruderal, mostly in lowlands. Weaker similarities were also observed with the south European subalpine taxa of the group of S.vallesia L. of section Auriculatae (Boiss.) Schischk., though these are not likely to reflect a real systematic affinity.

Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Survivability of the lichen Xanthoria parietina in simulated Martian environmental conditions.

Xanthoria parietina (L.) Th. Fr. is a widely spread foliose lichen showing high tolerance against UV-radiation thanks to parietin, a secondary lichen substance. We exposed samples of X. parietina under simulated Martian conditions for 30 days to explore its survivability. The lichen's vitality was monitored via chlorophyll a fluorescence that gives an indication for active light reaction of photosynthesis, performing in situ and after-treatment analyses. Raman spectroscopy and TEM were used to evaluate carotenoid preservation and possible variations in the photobiont's ultrastructure respectively. Significant differences in the photo-efficiency between UV irradiated samples and dark-kept samples were observed. Fluorescence values correlated with temperature and humidity day-night cycles. The photo-efficiency recovery showed that UV irradiation caused significant effects on the photosynthetic light reaction. Raman spectroscopy showed that the carotenoid signal from UV exposed samples decreased significantly after the exposure. TEM observations confirmed that UV exposed samples were the most affected by the treatment, showing chloroplastidial disorganization in photobionts' cells. Overall, X. parietina was able to survive the simulated Mars conditions, and for this reason it may be considered as a candidate for space long-term space exposure and evaluations of the parietin photodegradability.

Fatty acid acylated peptide therapeutics: discovery of omega-n oxidation of the lipid chain as a novel metabolic pathway in preclinical species.

We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.

Characterization of a new potent and long-lasting single chain peptide agonist of RXFP1 in cells and in vivo translational models.

Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.

Discovery of MK-1462: GLP-1 and Glucagon Receptor Dual Agonist for the Treatment of Obesity and Diabetes.

Peptide-based analogues of the gut-derived incretin hormone, glucagon-like peptide 1 (GLP1), stimulate insulin secretion in a glucose-dependent manner. Currently marketed GLP1 receptor (GLP1R) agonists are safe and effective in the management of Type 2 diabetes but often offer only modest weight loss. This has prompted the search for safe and effective alternatives to enhance the weight loss component of these treatments. We have demonstrated that concomitant activation GLP1R and the glucagon receptor (GCGR) can improve glucose metabolism and provide superior weight loss when compared to selective GLP1R agonism in preclinical species. This paper will highlight chemistry structure-activity relationship optimization and summarize in vivo efficacy studies toward the discovery of a once daily balanced dual agonist 12 (MK-1462), which was advanced into clinical trials.

Discovery of Insulin/GLP-1/Glucagon Triagonists for the Treatment of Diabetes and Obesity.

The combination of insulin and incretin-based therapies has emerged as a potential promising tactic for the treatment of diabetes. Here we report the first example of a unimolecular triagonist to simultaneously target insulin, GLP-1, and glucagon receptors, aiming for better glycemic control and superior weight loss. The strategy for constructing such a unimolecular triagonist is the conjugation of the insulin moiety and GLP-1R/GCGR coagonist peptide via alkyne-azide click chemistry. Two tractable series differentiated by insulin conjugation sites, B1F and B29K, were identified. Triagonist 13 prepared through the conjugation at insulin B1F and position 24 of GLP-1R/GCGR coagonist exhibited insulin activity comparable to that of insulin degludec and potent and balanced GLP-1R and GCGR activities. Pharmacokinetic profiles of 13 in both rat and minipig were also discussed.

SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).

Characterization of Blood Mucosal-Associated Invariant T Cells in Patients With Axial Spondyloarthritis and of Resident Mucosal-Associated Invariant T Cells From the Axial Entheses of Non-Axial Spondyloarthritis Control Patients.

OBJECTIVE: The importance of interleukin-17A (IL-17A) in the pathogenesis of axial spondyloarthritis (SpA) has been demonstrated by the success of IL-17A blockade. However, the nature of the cell populations that produce this important proinflammatory cytokine remains poorly defined. We undertook this study to characterize the major IL-17A-producing blood cell populations in the peripheral blood of patients with axial SpA, with a focus on mucosal-associated invariant T (MAIT) cells, a population known to be capable of producing IL-17. METHODS: We evaluated IL-17A production from 5 sorted peripheral blood cell populations, namely, MAIT cells, γδ T cells, CD4+ T cells, CD8+ T cells, and neutrophils, before and after stimulation with phorbol myristate acetate, the calcium ionophore A23187, and ß-1,3-glucan. Expression of IL-17A transcripts and protein were determined using nCounter and ultra-sensitive Simoa technology, respectively. MAIT cells from the axial entheses of non-axial SpA control patients (n = 5) were further characterized using flow cytometric immunophenotyping and quantitative polymerase chain reaction, and the production of IL-17 was assessed following stimulation. RESULTS: On a per-cell basis, MAIT cells from peripheral blood produced the most IL-17A compared to CD4+ T cells (P < 0.01), CD8+ T cells (P < 0.0001), and γδ T cells (P < 0.0001). IL-17A was not produced by neutrophils. Gene expression analysis also revealed significantly higher expression of IL17A and IL23R in MAIT cells. Stimulation of peripheral blood MAIT cells with anti-CD3/CD28 and IL-7 and/or IL-18 induced strong expression of IL17F. MAIT cells were present in the normal, unaffected entheses of control patients who did not have axial SpA and showed elevated AHR, JAK1, STAT4, and TGFB1 transcript expression with inducible IL-17A protein. IL-18 protein expression was evident in spinal enthesis digests. CONCLUSION: Both peripheral blood MAIT cells and resident MAIT cells in normal axial entheses contribute to the production of IL-17 and may play important roles in the pathogenesis of axial SpA.

Microclimatic Alteration after Logging Affects the Growth of the Endangered Lichen Lobaria pulmonaria.

Microclimatic conditions are important in determining lichen distribution at small scale, and may determine whether the species persist when the surrounding environmental conditions have drastically changed. This is the case with forest management, since a sudden variation of microclimatic conditions (increase of solar radiation, temperature, wind and a reduction of humidity) may occur after logging. In this study, the combined effect of forest logging and microclimatic conditions on the growth probabilities and growth rates of the model species Lobaria pulmonaria was assessed in mixed oak stands. To this purpose, 800 fragments of L. pulmonaria (<1 cm) were transplanted in logged and unlogged stands for two years. Young and adult fragments were positioned on Turkey oak boles according to distance from the ground (100 and 50 cm) and aspect (north and south). The results, evaluated by generalized linear mixed models on a yearly basis, highlighted differences in growth-particularly on isolated trees in the logged stand. South-exposed samples in the logged stand showed a low probability of growth, while samples transplanted north in the unlogged stand showed higher growth probabilities. However, the highest annual growth coefficients corresponded to south-exposed samples 50 cm from the ground in the unlogged stand. In general, higher growth rates were observed in young thallus fragments when compared with adult ones. Beyond confirming the importance of microclimate for lichen ecology, these results could be implemented in conservation actions to preserve L. pulmonaria populations in logged forests.

Impact of microplastics on growth, photosynthesis and essential elements in Cucurbita pepo L.

In this study, Cucurbita pepo L., one of the most cultivated, consumed and economically important crop worldwide, was used as model plant to test the toxic effects of the four most abundant microplastics identified in contaminated soils, i.e. polypropylene (PP), polyethylene (PE), polyvinylchloride (PVC), and polyethyleneterephthalate (PET). Cucurbita plants were grown in pots with increasing concentrations of the microplastics, then plant biometry, photosynthetic parameters and ionome of treated vs. untreated samples were compared to evaluate the toxicity of each plastic. All the pollutants impaired root and, especially, shoot growth. Specific and concentration-dependant effects of the different microplastics were found, including reduction in leaf size, chlorophyll content and photosynthetic efficiency, as well as changes in the micro- and macro-elemental profile. Among all the microplastics, PVC was identified as the most toxic and PE as the less toxic material. PVC decreased the dimensions of the leaf lamina, the values of the photosynthetic performance index and the plant iron concentration to a higher extent in respect to the other treatments. Microplastic toxicity exerted on the growth of C. pepo raises concerns about possible yield and economic loss, as well as for risks of a possible transfer into the food chain.

Expectations, experiences and preferences of patients and physicians in the informed consent process for clinical trials in oncology.

PURPOSE: The aim of the present study was to explore (1) informed consent (IC) representations, level of understanding, needs, and factors that influence the willingness of cancer patients to participate in randomized controlled trials (RCTs) (phase I) and (2) representations, experiences, and critical issues of physicians involved in the same process (phase II). METHODS: Semi-structured interviews were conducted with 20 cancer patients who had been asked to enroll in a phase II/III RCT (phase I). Two focus groups were conducted with 13 physicians enrolled in the same process (phase II). The content produced was analyzed through a thematic analysis. RESULTS: The themes that emerged in the first phase I were grouped into six categories: IC representation, randomization, experimentation, meeting with the physician, factors that influence the willingness to participate, and trial participants' needs. The themes emerged in the phase II were grouped into four: IC representation, critical issues of the IC, relationship, and recruitment of trial participants. Each theme is articulated into sub-themes and deeply discussed. CONCLUSION: This study highlights (1) the gap between what is ethically demanded in a RCT consultation and the reality of the situation and (2) the difference in perceptions between patients and physicians with reference to the meaning, objectives, and level of understanding of IC.

Guiding Chemically Synthesized Peptide Drug Lead Optimization by Derisking Mast Cell Degranulation-Related Toxicities of a NaV1.7 Peptide Inhibitor.

Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.