Assessment of the immunomodulatory properties of the probiotic strain Lactobacillus paracasei K5 in vitro and in vivo

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1α (IL-1α), ΙL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies

Development of BromoTag:A “Bump-&-Hole”-PROTAC system to induce potent, rapid, and selective degradation of tagged target proteins

Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure-activity relationships of our bump-and-hole-PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells and in vivo.</p

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is up-regulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including four endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are inefficiently phosphorylated by Akt as they possess an n + 1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.</p

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson s disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35 [D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.</p

真空紫外・軟X線領域の新しい光物性理論(講義,講義の報告,1985年度物性若手夏の学校報告)

この論文は国立情報学研究所の電子図書館事業により電子化されました

Assessment of PKC-dependent activation of LRRK1 in vitro v1

We describe a non-radioactive assay that we deploy for analysing the kinase activity of recombinant LRRK1 following in vitro activation by Protein kinase C (PKC) isoforms. This assay can also be used to analyse the effect of PKC on LRRK1 immunoprecipitated from cells. </p

Study of molecular markers in ovarian cancer

Ovarian cancer is one of the most common types of cancer and the leading cause of death due to gynecological malignancies. It is well established that dense CD8+ T-lymphocyte infiltration within the ovarian tumor masses correlates with favorable prognosis and improved patient survival. Still, the genes and the underlying molecular mechanisms that govern the recruitment and infiltration of T- lymphocytes in ovarian cancer remain quite vague. The aim of the present thesis was to identify genes involved in T-lymphocyte infiltration and to investigate their prognostic value and their role in the recruitment of lymphocytes in early stage ovarian cancer. In an attempt to pinpoint genes involved in CD8+ T-lymphocyte recruitment at the early stages of the disease, we focused in stage I and II ovarian tumors. To this end, a fluorescent Differential Display approach was utilized and 128 genes over-expressed in ovarian tumors with high numbers of CD8+ T-lymphocytes were identified. The expression of a significant number of genes was further evaluated with qPCR in a cohort of 38 early-stage ovarian cancer patients and a significant positive correlation of the mRNA levels of CXCL9, SMARCE1, MEIS1, GPC6, TMEM132D and JAG1 with the expression levels of CD8A, a marker of CD8+ T-lymphocyte infiltration, was documented. Importantly, forced over-expression of the chromatin remodeler SMARCE1 in SKOV3 ovarian cancer cells after transfection enhanced the expression of the chemokines IL8, RANTES and MIP1B and triggered the chemotaxis of CD8+ T-lymphocytes in vitro. Similarly, over-expression of the transcription factor MEIS1 in SKOV3 cells resulted in secretion of the chemokines IL8, GROα, RANTES, MIP1B, PARC and NAP2 and enhanced chemotaxis of CD8+ T-lymphocytes. Furthermore, MEIS1 was able to bind to the above chemokine promoters both in vitro and in vivo as found by Electrophoretic Mobility Shift Assays and Chromatin Immunoprecipitation. Finally, in order to evaluate the prognostic value of the genes identified with the fluorescent Differential Display, a retrospective clinical analysis of the survival of the early-stage ovarian cancer patients was performed. The expression of GPC6 and TMEM132D correlated with the overall survival of the patients and thus the two genes could serve as prognostic markers in early stage ovarian cancer. To sum up, in the present thesis several genes that could be used as markers of CD8+ T-lymphocyte infiltration and with prognostic value in early stage ovarian cancer were identified. Furthermore, two novel mechanisms regulating CD8+ T-lymphocyte recruitment at the tumor masses were demonstrated.O καρκίνος των ωοθηκών είναι μία από τις πιο κοινές μορφές καρκίνου και η σημαντικότερη αιτία θανάτου από γυναικολογικές κακοήθειες. Σύμφωνα με πληθώρα μελετών, η διήθηση CD8+ T-λεμφοκυττάρων στους όγκους ωοθηκών αποτελεί θετικό προγνωστικό δείκτη και συσχετίζεται με αυξημένη επιβίωση των ασθενών. Ωστόσο, δεν έχουν χαρακτηριστεί πλήρως τα γονίδια και οι υποκείμενοι μοριακοί μηχανισμοί που ρυθμίζουν τη στρατολόγηση των Τ-λεμφοκυττάρων στους συμπαγείς όγκους ωοθηκών. Στόχος, λοιπόν, της παρούσας διατριβής ήταν η ταυτοποίηση γονιδίων που σχετίζονται με τη διήθηση των λεμφοκυττάρων στον καρκίνο ωοθηκών και η μελέτη α) του ρόλου τους σε αυτή τη διαδικασία και β) της προγνωστικής τους αξίας. Για τον εντοπισμό γονιδίων που εμπλέκονται στις αρχικές φάσεις της διήθησης των CD8+ T-λεμφοκυττάρων, επικεντρωθήκαμε σε αρχικού σταδίου καρκινικούς όγκους ωοθηκών. Εφαρμόζοντας μια σύνθετη μέθοδο Διαφορικής Έκθεσης με φθορισμό ταυτοποιήσαμε 128 γονίδια που υπερεκφράζονταν σε όγκους με εκτεταμένη διήθηση CD8+ T-λεμφοκυττάρων. Για ένα σημαντικό αριθμό από αυτά η έκφραση επαληθεύθηκε με ποσοτική PCR σε 38 ασθενείς με καρκίνο ωοθηκών αρχικού σταδίου και παρατηρήθηκε μια θετική και στατιστικά σημαντική συσχέτιση των επιπέδων έκφρασης των γονιδίων CXCL9, SMARCE1, MEIS1, GPC6, TMEM132D, CST3 και JAG1 με τη διήθηση των CD8+ T-λεμφοκυττάρων. Μάλιστα, η υπερέκφραση του παράγοντα αναδιαμόρφωσης της χρωματίνης SMARCE1 σε κύτταρα της καρκινικής σειράς ωοθηκών SKOV3, ύστερα από διαμόλυνση, είχε ως αποτέλεσμα την επαγωγή της έκφρασης των χημειοκινών IL8, RANTES και MIP1B και προκαλούσε τον in vitro χημειοτακτισμό CD8+ T-λεμφοκυττάρων. Επίσης, με ανοσοφθορισμό βρέθηκε ότι ο μεταγραφικός παράγοντας MEIS1 εκφράζεται σε συγκεκριμένες περιοχές καρκινικών όγκων ωοθηκών με εκτεταμένη διήθηση λεμφοκυττάρων. Επιπλέον, η υπερέκφραση του MEIS1 σε κύτταρα της καρκινικής σειράς ωοθηκών SKOV3 είχε ως αποτέλεσμα την επαγωγή της έκφρασης των χημειοκινών IL8, GROα, RANTES, MIP1B, PARC και NAP2 και προκαλούσε τον χημειοτακτισμό CD8+ T-λεμφοκυττάρων in vitro. Ακόμη, με δοκιμές Μεταβολής Ηλεκτροφορητικής Κινητικότητας και Ανοσοκατακρήμνιση Χρωματίνης ο παράγοντας MEIS1 βρέθηκε ότι προσδένεται in vitro και in vivo στους υποκινητές των παραπάνω χημειοκινών, ενισχύοντας την πιθανότητα να ρυθμίζει άμεσα την έκφρασή τους. Τέλος, αναλύοντας την προγνωστική αξία των γονιδίων που ταυτοποιήθηκαν με τη μέθοδο Διαφορικής Έκθεσης, βρέθηκε ότι τα επίπεδα έκφρασης των GPC6 και TMEM132D συσχετίζονται σημαντικά με την ολική επιβίωση των ασθενών και αποτελούν θετικό προγνωστικό δείκτη στον καρκίνο ωοθηκών αρχικού σταδίου. Συμπερασματικά, στην παρούσα μελέτη ταυτοποιήθηκαν γονίδια που θα μπορούσαν χρησιμοποιηθούν ως δείκτες πρόγνωσης και διήθησης CD8+ T-λεμφοκυττάρων στον καρκίνο των ωοθηκών και προσδιορίστηκαν δύο νέοι μηχανισμοί ρύθμισης αυτής της διήθησης