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Background Pathogen reduction technologies (PRT) based on nucleic-acid damaging chemicals and/or irradiation are increasingly being used to increase safety of blood components against emerging pathogens, such as convalescent plasma in the ongoing COVID-19 pandemic. Current methods for PRT validation are limited by the resources available to the blood component manufacturer, and quality control rely over pathogen spiking and hence invariably require sacrifice of the tested blood units: quantitative real-time PCR is the current pathogen detection method but, due to the high likelihood of detecting nonviable fragments, requires downstream pathogen culture. We propose here a new molecular validation of PRT based on the highly prevalent human symbiont torquetenovirus (TTV) and rolling circle amplification (RCA). Materials and methods Serial apheresis plasma donations were tested for TTV before and after inactivation with Intercept® PRT using real-time quantitative PCR (conventional validation), RCA followed by real-time PCR (our validation), and reverse PCR (for cross-validation). Results While only 20% of inactivated units showed significant decrease in TTV viral load using real-time qPCR, all donations tested with RCA followed by real-time PCR showed TTV reductions. As further validation, 2 units were additionally tested with reverse PCR, which confirmed absence of entire circular genomes. Discussion We have described and validated a conservative and easy-to-setup protocol for molecular validation of PRT based on RCA and real-time PCR for TTV.

Purpose Lax eyelid condition (LEC) and floppy eyelid syndrome (FES) represent two distinct conditions which have been associated with several ocular and systemic comorbidities. The main aim of this systematic review and meta-analysis is to explore the available literature to estimate the prevalence rate of LEC and FES in obstructive sleep apnea (OSA). Methods The protocol of this systematic review and meta-analysis has been registered in PROSPERO. Four electronic databases (PubMed/MEDLINE, Google Scholar, Cochrane Library, Web of Science) were searched from inception to December 24, 2021. A random intercept logistic regression model was carried out for the analysis of overall proportions. Odds ratio and mean difference were reported as measures of the effect size in the presence of binary and continuous outcomes, respectively. The estimated numbers of LEC/FES patients in OSA were calculated by multiplying the prevalence rate determined by our random-effects model and the corresponding Benjafield et al.’s population prospect. Results We included 11 studies comprising 1225 OSA patients of whom 431 and 153 affected by LEC and FES, respectively. Our model estimated a pooled prevalence rate for LEC and FES in OSA patients of 40.2% (95%CI: 28.6–53.1%) and of 22.4% (95%CI: 13.8–34.2%), respectively. The number of LEC/FES affected individuals among OSA patients is expected to peak up to 376 and to 210 million, respectively. OSA patients appeared to have a 3.4 (95%CI: 2.2–5.2) and a 3.0 (95%CI: 1.7–5.5) increased risk of developing LEC and FES than the healthy counterpart. Conclusion Prevalence of LEC and FES is higher in OSA-affected patients compared to controls. More studies are warranted to investigate the mechanisms leading to the development of LEC and/or FES in OSA patients, as well as the feasibility of the adoption of these clinical findings as screening tools for OSA.

To accurately time motor responses when intercepting falling balls we rely on an internal model of gravity. However, whether and how such a model is also used to estimate the spatial location of interception is still an open question. Here we addressed this issue by asking 25 participants to intercept balls projected from a fixed location 6 m in front of them and approaching along trajectories with different arrival locations, flight durations, and gravity accelerations (0 g and 1 g). The trajectories were displayed in an immersive virtual reality system with a wide field of view. Participants intercepted approaching balls with a racket, and they were free to choose the time and place of interception. We found that participants often achieved a better performance with 1 g than 0 g balls. Moreover, the interception points were distributed along the direction of a 1 g path for both 1 g and 0 g balls. In the latter case, interceptions tended to cluster on the upper half of the racket, indicating that participants aimed at a lower position than the actual 0 g path. These results suggest that an internal model of gravity was probably used in predicting the interception locations. However, we found that the difference in performance between 1 g and 0 g balls was modulated by flight duration, the difference being larger for faster balls. In addition, the number of peaks in the hand speed profiles increased with flight duration, suggesting that visual information was used to adjust the motor response, correcting the prediction to some extent. NEW & NOTEWORTHY Here we show that an internal model of gravity plays a key role in predicting where to intercept a fast-moving target. Participants also assumed an accelerated motion when intercepting balls approaching in a virtual environment at constant velocity. We also show that the role of visual information in guiding interceptive movement increases when more time is available.

Aims and background The aim of our study was to estimate the incidence of second primary tumors among breast cancer patients from the Division of Oncology of Andosilla Hospital (Viterbo, Italy). In particular, we studied the relationship between breast and colorectal cancer. Methods and Study Design Eligible women were those with primary invasive breast cancer who had been treated and/or followed up at our division. We compared our data with those reported in the literature. Results Of 114 women with breast cancer, 21 (18.5%) developed multiple primary cancers, with colorectal cancer accounting for a quarter. We found a higher incidence of colorectal cancer than reported in the literature (5% vs 0.66–1%). At the moment we are not able to explain this difference. Conclusions Quite a few studies reported a link between breast and colorectal cancer, but the magnitude of the risk (standardized incidence ratio 1.3–2) does not justify a screening program for colorectal disease in breast cancer patients. However, considering that the risk is small but not negligible – along with the high incidence of colorectal adenomas described in breast cancer patients and the possible existence of common risk factors – we invite clinicians not to neglect the possibility of a colorectal cancer diagnosis in women who have had breast cancer.

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [ 125 I ]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [ 125 I ]ET-1 in a biphasic manner, in contrast to an ET B -selective agonist, IRL-1620, that was ineffective. The ET B -selective antagonist, BQ-788, inhibited [ 125 I ]ET-1 binding in a monophasic manner and with low potency. An ET A -selective antagonist, BQ-123, competed [ 125 I ]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET A and -ET B antibodies confirmed a predominant expression of the ET A receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1 mM CaCl 2 . Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-β inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration–response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca 2+ resulted in a shift to the right of the ET-1 concentration–response curve. Both the L-type voltage-operated Ca 2+ channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET A receptors are expressed and functionally coupled to rise of [Ca 2+ ] i in H9c2 cardiomyoblasts. ET-1-induced [Ca 2+ ] i increase is triggered by Ca 2+ release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca 2+ channels and ryanodine receptors participate in sustaining the Ca 2+ response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca 2+ ] i increase.

Endothelins exert their physiological effects through interaction with cell surface receptors that are members of the G-protein-coupled receptor family. The endothelin receptor subtype B (ET(B) receptor) is abundantly expressed in rat cerebellum. Since agonist binding to G-protein-coupled receptors may be modulated by cations and guanine nucleotides, we investigated the effects of cations and guanosine 5'-O-(2-thiotriphosphate) (GTPgammaS) on 125I-endothelin-1 (125I-ET-1) binding to rat cerebellar membranes. Both Na+ and Mg2+-stimulated 125I-ET-1 binding causing an increase in receptor affinity for the agonist. While the effect of the divalent cation was evident at relatively low concentrations (5-10 mM), the stimulatory activity of the monovalent cation appeared at relatively high concentrations (50 mM). Additive activities of 25-50 mM NaCl and 1 mM MgCl2 suggested that monovalent and divalent cations increased receptor affinity for ET-1 by different mechanisms. In the presence of 5 mM MgCl2, 50 mM NaCl caused an additional modest reduction of the Kd value. Whereas 5 mM MgCl2 affected the displacement curves of both ET-3 and suc-[Glu9, Ala11,15]-endothelin-1 (8-21) (IRL 1620), the influence of 50 mM NaCl on these curves was less substantial. All together, these results suggest that modulation of receptor affinity by NaCl depends on the nature of the displacing agonist. In the presence of 5 mM MgCl2 or 50 mM NaCl, a partial regulation of 125I-ET-1 binding by GTPgammaS was detectable, while in the absence of cations no GTPgammaS-dependent inhibition was evident.

The pharmacological properties of endothelin receptors (ETR) were investigated in guinea-pig bronchus by comparing binding and functional results. In binding assays, both the ETB agonists, endothelin-3 (ET-3) and N-suc-[Glu9,Ala11,15]ET-1(8–21) (IRL 1620), and the antagonist, N-cis-2,6-dimethylpiperidinocarbonyl-L-γ-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (BQ 788), showed biphasic inhibition curves of [125I]-endothelin-1 (ET-1) binding to bronchus membranes prepared from intact or epithelium-deprived tissue. IRL 1620 did not completely displace specifically [125I]-ET-1 bound to these tissue preparations. In the presence of the ETA-selective antagonist, cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu) (BQ 123, 1 μM), IRL 1620 displacement curves were shallow but a complete inhibition was reached at a concentration of 1 μM. Both curves were better represented by two-site models. In addition, BQ 788 competition curves became monophasic when binding experiments were performed in the presence of 1 μM BQ 123. The non-selective agonist, ET-1, and BQ 123 inhibited [125I]-ET binding to bronchus membranes in dose-dependent fashions with monophasic curves. The contracting activity of IRL 1620 (0.55 nM–1.6 μM) was tested on multiple-ring bronchial preparations pretreated with peptidase and cyclo-oxygenase inhibitors. BQ 788 shifted IRL1620 concentration-response curves to the right while BQ 123 did not influence bronchial responsiveness. In addition, a potentiation of the maximal response to the agonist was observed in BQ 788 treated bronchial rings. This effect was abolished by tissue pretreatment with Nω-nitro-L-argininemethylester (L-NAME) or epithelium removal but not by pretreatment with atropine or iberiotoxin. Our results demonstrate that guinea-pig bronchus contains two populations of ETB receptors with different affinities for the ETB-selective agonist, IRL 1620. One ETB receptor population appears to activate bronchial muscle contraction while another on epithelial cells causes muscle relaxation through the release of nitric oxide (NO). Keywords: Endothelin receptors, ETB receptors, endothelins, guinea-pig bronchus Introduction Endothelins (ETs)1, a family of three closely related peptide hormones, endothelin-1, -2, and -3 (ET-1, ET-2, and ET-3), were originally recognized as potent modulators of vascular functions participating in both vasoconstriction and vasodilatation. Thereafter, the presence of ETs was demonstrated in several other tissues of different species including humans, where they exert various physiological effects (for a review see: Rubanyi & Polokoff, 1994; Webb et al., 1998). All activities of ETs are mediated through the interaction with specific cell surface receptors (for a review see: Bax & Saxena, 1994; Ohlstein et al., 1996; Pollock et al., 1995). Two ETR subtypes, termed ETA and ETB, have been identified, cloned and expressed from rat (Lin et al., 1991; Sakurai et al., 1990; Sugiura et al., 1989), bovine (Arai et al., 1990; Saito et al., 1991), and human tissues (Adachi et al., 1991; Cyr et al., 1991; Ogawa et al., 1991; Sakamoto et al., 1991). Both receptor subtypes belong to the large receptor family characterized by seven transmembrane-spanning regions which regulate distinct intracellular signalling pathways through the activation of G proteins. The two subtypes can be distinguished by different rank orders of affinity toward the three ET isopeptides. The ETA receptor binds ET-1, ET-2 and ET-3 with decreasing affinities whereas the ETB receptor shows a similar affinity for all three isopeptides. An additional classification of both receptor subtypes as ETA1, ETA2, ETB1, and ETB2 has been proposed on the basis of their affinities for agonists or antagonists (Karaki et al., 1994; Sudjarwo et al., 1994). The ETA receptor mediates contraction of arterial smooth muscles, while the ETB receptor exhibits either contractile or relaxing effects depending on the vascular bed examined (Pollock et al., 1995). Selective activation of ETB receptors on endothelial cells causes relaxation of rat aorta through the release of nitric oxide (Karaki et al., 1993). However, activation of ETB receptors on venous smooth muscles gives rise to vasoconstriction (Moreland et al., 1994). ETs modulate airway activities of different species including humans (Hay et al., 1993; Knott et al., 1995). These peptides are potent constrictors of airways both in vivo and in vitro (Hay et al., 1993; Touvay et al., 1990). In guinea-pig trachea and bronchus, the contraction induced by ET-1 appears to involve a mixed population of ETRs (Hay et al., 1993). In addition, regional differences of ETR subtype distribution seem to exist in guinea-pig airways, with bronchus expressing mainly ETB receptor subtypes (Hay et al., 1993). In guinea-pig trachea, functional (Battistini et al., 1993) and autoradiographic studies (Tschirhart et al., 1991) have shown the presence of ETRs on the epithelium. Functional data reported by Battistini et al. (1993) have also indicated that in guinea-pig trachea, ET-1 activates ETA receptors on epithelial cells, inducing the release of prostanoids, while airway contraction is mediated via ETB receptors probably present on smooth muscles. Recently, it has been suggested (Yoneyama et al., 1995b) that ETs modulate the airway tone by acting not only directly on airway smooth muscle, but also on the cholinergic nerve, to modulate acetylcholine (ACh) release. The pathophysiological role of ETs in asthma and other respiratory disorders has received some attention (Hay et al., 1993). The presence of ETB receptors in guinea-pig and human bronchus mediating smooth muscle contraction is well established (Battistini et al., 1994; Hay et al., 1993; 1998; Hay & Luttman, 1997). However, in guinea-pig this receptor population has not been characterized for its biochemical and pharmacological features. Two subtypes of ETA and ETB receptors seem to mediate smooth muscle contraction of rabbit trachea (Yoneyama et al., 1995a). Similarly, the ETB receptor population in guinea-pig bronchus cannot be homogeneous. As guinea-pig bronchus is frequently used as an experimental model to test the activity of new anti-asthmatic drugs, characterization of its ETB receptor population is pivotal for elucidating the pathophysiological significance of ETs in asthma. Thus, we investigated the biochemical and pharmacological properties of ETRs in guinea-pig bronchus using selective agonist and antagonist ligands.

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.

Increased expression of urokinase (uPA), a member of the serine protease family, is an effector of metastatic cascade and has been reported in various malignancies, including breast cancer. uPA overexpression in cancer tissues was correlated with a more aggressive phenotype and it is considered a strong and independent unfavorable prognostic factor in breast cancer. Using real-time PCR assay, we analyzed uPA expression of malignant and benign breast nodular lesions versus healthy tissues (normal breast and lymphocytes). We found that besides breast cancer nodule, normal mammary gland and lymphocytes overexpressed uPA too. Tissues obtained from women with benign lesions expressed homogeneous and lower uPA. In conclusion, although uPA overexpression is typical of cancer tissues, it could be considered as a feature of the whole organism affected by cancer. On the basis of these first results, uPA could be considered for further studies as a possible useful therapeutic target in breast cancer.

This study demonstrates that, in the nasal respiratory mucosa of patients with vasomotor rhinitis, oxidative stress following peroxynitrite formation is confined to the respiratory epithelium. This suggests that the role of peroxynitrite in vasomotor rhinitis differs from its role in other diseases of the respiratory tract. The results of this study also support the concept that different pathogenetic mechanisms are probably involved in vasomotor rhinitis.Previous studies indicated that nitric oxide (NO) is involved in the pathogenesis of vasomotor rhinitis, strong expression of NO synthase being detected in the smooth muscle cells of the cavernous sinuses and in the respiratory epithelium. However, most adverse effects of high levels of NO originate from the reaction of NO with superoxide anions to form peroxynitrite. Therefore, in this study we evaluated the involvement of peroxynitrite in the pathogenesis of vasomotor rhinitis.Sites of peroxynitrite formation were identified by immunolabelling for 3-nitrotyrosine (3NT), its footprint in tissues. Samples of nasal mucosa were obtained from vasomotor rhinitis patients and from control subjects who had undergone corrective surgery of the nasal septum. All samples were obtained by reduction of the inferior turbinate.Examination of specimens from vasomotor rhinitis patients revealed that 3NT is absent in epithelium with a normal appearance, cells of the subepithelial connective tissue, the glands and the blood vessels, including the cavernous sinuses. In contrast, intense 3NT immunolabelling was found in the disrupted respiratory epithelium. 3NT was not present in any of the specimens from control subjects.

The change of selectivity and the induction of antagonism by the insertion of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the second position of several opioid peptides have led to the interpretation of Tyr-Tic as a specific message domain for δ-opioid antagonists and to the discovery of dipeptides with substantial opioid activity. Selectivity and activity increase enormously when Tyr is substituted by 2′,6′-dimethyl tyrosine (Dmt), hinting that the side chain of Dmt fits a hydrophobic cavity of the receptor very tightly and precisely. We have investigated the specificity of this fit by systematic changes of the substituents on the aromatic ring of Tyr. Mono- and disubstitutions different from 2′,6′- invariably lead to catastrophic decreases of activity. The only substitution compatible with retention of substantial antagonism is 2′-methyl. An analysis of the conformational properties of all analogues reveals that substitutions do not affect the global shape of the molecule significantly. Accordingly, it is possible to use the shape of the different side chains to map the hydrophobic cavity of the receptor. The resulting complementary image is funnel shaped. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.