Preprotein mature domains contain translocase targeting signals that are essential for secretion

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion

Έκβαση βαρέως πασχόντων COVID-19 ασθενών που λαμβάνουν υψηλή και χαμηλή δόση δεξαμεθαζόνης σε συνδυασμό με anti-IL6 μονοκλωνικό αντίσωμα (Tocilizumab) στη Μονάδα Εντατικής Θεραπείας

Εισαγωγή: Στη σοβαρή νόσο COVID -19 έχει καθιερωθεί η χορήγηση δεξαμεθαζόνης. Η χορήγηση υψηλότερης δόσης κορτικοστεροειδών ή/και Tocilizumab είναι υπό διερεύνηση. Σκοπός: Να αξιολογηθούν οι διαφορές ως προς την έκβαση και τις επιπλοκές των διαφορετικών δόσεων κορτικοστεροειδών (υψηλή δόση vs συνήθους δόσης) συν/πλην Tocilizumab. Υλικό και Μέθοδος: Πραγματοποιήθηκε μία αναδρομική μελέτη στη ΜΕΘ της Α’ Πανεπιστημιακής Πνευμονολογικής της Ιατρικής Σχολής ΕΚΠΑ το διάστημα 1/9/2020-1/9/2021 σε βαρέως πάσχοντες COVID -19 ασθενείς. Έγινε καταγραφή επιδημιολογικών στοιχείων, συννοσηροτήτων, χαρακτηριστικών της νόσησης, της έκβασης, καθώς και της λήψης κορτικοστεροειδών συν / πλην Tocilizumab. Ως συνήθης δόση ορίστηκε η δεξαμεθαζόνη 6mg και ως υψηλή οποιοδήποτε σχήμα μεγαλύτερης δόσης ημερησίως. Αποτελέσματα: Συμπεριελήφθησαν 300 ασθενείς. Από τη μονοπαραγοντική ανάλυση, οι ασθενείς που έλαβαν υψηλή δόση δεξαμεθαζόνης είχαν στατιστικά σημαντική αύξηση της πιθανότητας διασωλήνωσης στη ΜΕΘ, αύξηση των επιπλοκών (λοιμώξεις, βαρότραυμα, θρομβοεμβολικό επεισόδιο), καθώς και αυξημένη διάρκεια υποστήριξης από μηχανικό αερισμό και νοσηλείας στη ΜΕΘ και το νοσοκομείο. Από τις καμπύλες Kaplan Meier οι ασθενείς που έλαβαν υψηλή δόση δεξαμεθαζόνης είχαν καλύτερη επιβίωση στις 28 ημέρες ενώ στη συνολική επιβίωση δεν υπήρχε διαφορά. Η μη διαφορά της συνολικής επιβίωσης διατηρείται στην πολυπαραγοντική ανάλυση. Επίσης, από την πολυπαραγοντική ανάλυση δεν παρατηρήθηκαν στατιστικά σημαντικές διαφορές ως προς τις ημέρες μηχανικού αερισμού. Από τη σύγκριση της ομάδας των ασθενών που έλαβαν υψηλή δόση δεξαμεθαζόνης με Tocilizumab με την ομάδα των ασθενών που έλαβαν υψηλή δόση κορτικοστεροειδών χωρίς Tocilizumab δε διαπιστώθηκαν διαφορές ως προς την έκβαση (συνολική επιβίωση, ημέρες μηχανικού αερισμού), αλλά παρατηρήθηκε αύξηση των λοιμώξεων στην πρώτη ομάδα. Συμπεράσματα: H υψηλή δόση δεξαμεθαζόνης φαίνεται ότι έχει πιθανώς θετική επίδραση στην επιβίωση των ασθενών, αλλά αρνητική στην ανάπτυξη επιπλοκών. Ως προς τη συγχορήγηση Tocilizumab ή όχι, δε διαπιστώθηκε στατιστικά σημαντική διαφορά στην έκβαση.Introduction: Dexamethasone is the mainstay of treatment for severe COVID -19. Higher dosage of dexamethasone and/or Tocilizumab are still under investigation. Aim: To assess differences in outcome and complications of different doses of corticosteroids (high vs standard dose) and/or Tocilizumab. Material and Methods: A retrospective study was carried out on COVID-19 patients hospitalized in the ICU of the 1st Respiratory Medicine Department of National and Kapodistrian University of Athens during the period 1/9/2020-1/9/2021. Patient and disease characteristics, comorbidities, outcomes, corticosteroid dosage and Tocilizumab administration were recorded. Standard dose was defined as 6mg of dexamethasone for 10 days and high dose as any higher dose regimen per day. Results: 300 patients were recruited in the study. On univariate analysis, patients who received high dose dexamethasone had a statistically significant increase in the probability of intubation in the ICU, complications (infections, barotrauma, thromboembolic events) as well as increased length of mechanical ventilation and increased length of stay in ICU and hospital. Kaplan Meier curves showed that patients who received high-dose dexamethasone had better survival at 28 days, while overall survival did not differ at Kaplan Meier curves or multivariate analysis. Also, multivariate analysis showed no statistically significant differences in terms of days of mechanical ventilation between the groups. Patients receiving high-dose dexamethasone with Tocilizumab and patients receiving high-dose corticosteroids without Tocilizumab had no statistically significant differences in outcome (overall survival, days of mechanical ventilation), but the first group had an increased incidence of infections. Conclusion: High-dose dexamethasone seems to have a possibly positive effect on patient survival, but a negative effect on the development of complications. Regarding the co-administration of Tocilizumab or not, no statistically significant difference on the outcome was found

Καρκίνος του μαστού κατά την κύηση

Στην παρούσα διπλωματική εργασία εξετάζεται εκτενώς ο μαστός καθώς και οι παθήσεις αυτού, όπως ο καρκίνος. Ο μαστός είναι ένα εξαιρετικά ευαίσθητο και πολύπλοκο όργανο γι’ αυτό η δομή και η λειτουργία του μπορούν να επηρεαστούν από ενδογενείς και εξωγενείς παράγοντες, όπως το κληρονομικό ιστορικό, το περιβάλλον διαβίωσης, διατροφή και συνήθειες του ατόμου κλπ. Οι απεικονιστικές μέθοδοι σε συνδυασμό με την κλινική εξέταση, συμβάλλουν στην άμεση διάγνωση και θεραπεία της εκάστοτε πάθησης. Ανάλογα πάντα με τον βαθμό σοβαρότητας και εξέλιξης της βλάβης, επιλέγεται η κατάλληλη θεραπεία, η οποία θα είναι είτε φαρμακευτική, είτε συντηρητική είτε χειρουργική. Αναφέρονται επίσης και λύσεις για τη διατήρηση της γονιμότητας της γυναίκας πριν από οποιαδήποτε θεραπεία. Σύμφωνα με νέες μελέτες η εξέλιξη στην θεραπεία του καρκίνου μαστού είναι η ανοσοθεραπεία η οποία έχει τις λιγότερο δυνατές συνέπειες στον οργανισμό της γυναίκας καθώς και εκπληκτικά αποτελέσματα έναντι του καρκίνου. Ο συνδυασμός οποιασδήποτε πάθησης του μαστού, ιδιαίτερα του καρκίνου, με την κύηση δυσχεραίνει ακόμα περισσότερο την αντιμετώπισή του. Σε ο, τι αφορά την διάγνωση, οι εξετάσεις που μπορούν να γίνουν για την εκτίμηση της βλάβης του μαστού είναι περιορισμένες λόγω της υπάρχουσας κύησης. Ακόμα πιο περιορισμένες είναι οι επιλογές της θεραπείας λόγω ενδεχόμενης βλάβης του εμβρύου. Με τις όλο και περισσότερο εξελισσόμενες στον τομέα του καρκίνου έρευνες, πλέον η διάγνωση και η θεραπεία μπορεί να γίνει πιο εύκολα και γρήγορα χωρίς να κινδυνεύει το έμβρυο. Μην ξεχνάμε όμως ότι ο καρκίνος μαστού στην κύηση είναι μία πολύ δύσκολη κατάσταση διότι η γυναίκα εκτός από την ασθένειά της θα πρέπει να ανησυχεί και για το έμβρυό της. Για το λόγο αυτό θα πρέπει να έχει ψυχολογική στήριξη καθ’ όλη τη διάρκεια της κύησης αλλά και μετέπειτα.Regarding this dissertation, the breast is examined extensively as well as all of its diseases, such as cancer. The breast is an extremely sensitive and complicated organ that is why its structure and function can be affected by intrinsic and extrinsic factors, such as the hereditary background, the living environment, the nutrition and the daily habits of the person. All the essential tests, in combination with the clinical examination, contribute to the immediate diagnosis and the treatment of the specific disease. Suitable intervention must be chosen depending on the seriousness and the development of the damage, which is going to be either pharmaceutical, conventional or surgical. Moreover, there are some solutions referred to the preservation of woman’s fertility before any kind of treatment. According to new researches, the evolution in the cure of breast cancer is immunotherapy which has less possible effects to the organism of a woman and amazing results against cancer. The combination of any breast disease, especially the cancer one, with pregnancy makes its effectiveness even more difficult. As far as the diagnosis is concerned, the tests that can be runfor the evaluation of the damage of the breast are limited due to pregnancy. With all the more developed researches in the sector of cancer, both the diagnosis and the treatment can be done faster and easier without setting the embryo in danger. We should bear in mind though, that breast cancer throughout pregnancy is a very difficult situation to cope with, since the woman has to worry, besides her disease, about her baby as well. For this reason, she should have psychological support, not only throughout her pregnancy but after she gives birth as well

Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes

UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidulans, is a member of a large family of nucleobase-ascorbate transporters conserved in all domains of life. We have investigated structure-function relationships in UapA, by studying chimeric transporters and missense mutations, and showed that specific polar or charged an-Lino acid residues (E412, E414, Q449, N450, T457) on either side of an amphipathic alpha -helical transmembrane segment (TMS10) are critical for purine binding and transport. Here, the mutant Q449E, having no uric acid-xanthine transport activity at 25 degreesC, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine. All seven revertants were found to carry the mutation F569S within the last transmembrane segment (TMS14) of UapA. Further kinetic analysis of a selected suppressor showed that UapA-Q449E/F569S transports with high affinity (K-M values of 4-10 muM) xanthine, hypoxanthine and uracil. Uptake competition experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thioguanine, adenosine or ascorbic acid. A strain carrying mutation F569S by itself conserves high-capacity, high-affinity (K-M values of 1.5-15 muM), transport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compounds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10-L10) are critical for the uptake specificity and transport kinetics of UapA. (C) 2001 Academic Press

A novel-type substrate-selectivity filter and ER-exit determinants in the UapA purine transporter

We present a functional analysis of the last α-helical transmembrane segment (TMS12) of UapA, a uric acid-xanthine/H+ symporter in Aspergillus nidulans and member of the nucleobase-ascorbate transporter (NAT) family. First, we performed a systematic mutational analysis of residue F528, located in the middle of TMS12, which was known to be critical for UapA specificity. Substitution of F528 with non-aromatic amino acid residues (Ala, Thr, Ser, Gln, Asn) did not affect significantly the kinetics of UapA for its physiological substrates, but allowed high-capacity transport of several novel purines and pyrimidines. Allele-specific combinations of F528 substitutions with mutations in Q408, a residue involved in purine binding, led to an array of UapA molecules with different kinetic and specificity profiles. We propose that F528 plays the role of a novel-type selectivity filter, which, in conjunction with a distinct purine-binding site, control UapA-mediated substrate translocation. We further studied the role of TMS12 by analysing the effect of its precise deletion and chimeric molecules in which TMS12 was substituted with analogous domains from other NATs. The presence of any of the TMS12 tested was necessary for ER-exit while their specific amino acid composition affected the kinetics of chimeras. © 2005 Elsevier Ltd. All rights reserved

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and = O6, and to a lower extent with = O2. The E. coli homologue interacts principally with N3-H and = O2, and less strongly with N1-H and = O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with = O2, = O6 and = O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving = O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently