Leucine-rich-repeat-kinase 1 (LRRK1) and its homolog LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson's disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ∼3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the αC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.

Malus trilobata, is a rare tree species occurring in several small and disjunct populations in the eastern part of the Mediterranean basin. The main European population is found in the region of Evros (NE Greece) and is divided in five distinct subpopulations following the species geographical pattern. The genetic diversity of approximately the entire population (69 trees) was analyzed using nuclear microsatellite and random genomic markers. Polymorphism was discovered in 29 out of 45 genomic marker loci (64.44%), while for nuclear microsatellite markers, all three loci were polymorphic with an average of 3.75 alleles per locus. Our results unraveled a specific grouping pattern for both markers. Both genetic markers exhibited relatively low genetic diversity which is in accordance with the prevalent perception that species with fragmented distributions tend to have low genetic diversity, while the differentiation among individuals, revealed a patchy pattern among small groups of trees separated by roads, firebreaks or distance. These results indicate a high genetic fragmentation level for the main European population of M. trilobata, while the presence of roads, firebreaks, conifer plantations and agricultural land, seem to function as potential barriers to gene flow. Consequently, and since it is well-documented that bees hesitate to change foraging patches, as long as their food is abundant, the observed genetic differentiation patterns could be partially attributed to the foraging and flight behavior of bees, which are the main pollinators of the species. The low levels of available genetic diversity combined with the small overall population and repeated events of forest fires inside the M. trilobata distribution, perils the survival of the species and imposes the necessity for a thoroughly organized conservation strategy.

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.

Propolis is a resinous substance produced by bees that exhibits antimicrobial, immunostimulatory and antioxidant activity. Its use is common in functional foods, cosmetics and traditional medicine despite the fact that it demonstrates low extraction yields and inconsistency in non-toxic solvents. In this work, a new encapsulation and delivery system consisting of liposomes and cyclodextrins incorporating propolis polyphenols has been developed and characterized. The antioxidant, antimutagenic and antiaging properties of the system under normal and UVB-induced oxidative stress conditions were investigated in cultured skin cells and/or reconstituted skin model. Furthermore, the transcript accumulation for an array of genes involved in many skin-related processes was studied. The system exhibits significant polyphenol encapsulation efficiency, physicochemical stability as well as controlled release rate in appropriate conditions. The delivery system can retain the anti-mutagenic, anti-oxidative and anti-ageing effects of propolis polyphenols to levels similar and comparable to those of propolis methanolic extracts, making the system ideal for applications where non-toxic solvents are required and controlled release of the polyphenol content is desired.

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.

Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinson’s disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but 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 Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.### Competing Interest StatementThe authors have declared no competing interest.

In the present study, we aimed to examine the antioxidant, antiaging and photoprotective properties of Greek honey samples of various botanical and geographical origin. Ethyl-acetate extracts were used and the and the total phenolic/flavonoid content and antioxidant capacity were evaluated. Honey extracts were then studied for their cytoprotective properties against UVB-induced photodamage using human immortalized keratinocytes (HaCaT) and/or reconstituted human skin tissue models. Specifically, the cytotoxicity, oxidative status, DNA damage and gene expression levels of specific matrix metalloproteinases (MMPs) were examined. Overall, the treatment of HaCaT cells with honey extracts resulted in lower levels of DNA strand breaks and attenuated the decrease in cell viability following UVB exposure. Additionally, honey extracts significantly decreased the total protein carbonyl content of the irradiated cells, however, they had no significant effect on their total antioxidant status. Finally, the extracts alleviated the UVB-induced up-regulation of MMPs-3, -7 and -9 in a model of reconstituted skin tissue. In conclusion, honey extracts exhibited significant photoprotective and antiaging properties under UVB exposure conditions and thus could be further exploited as promising agents for developing novel and naturally-based, antiaging cosmeceutical products.

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.

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.

Probiotics are defined as live microorganisms that when administered in adequate amounts confer a health benefit to the host. Their positive supplementation outcomes on several gastrointestinal disorders are well defined. Nevertheless, their actions are not limited to the gut, but may also impart their beneficial effects at distant sites and organs. In this regard, in this review article we: (i) comprehensively describe the main mechanisms of action of probiotics at distant sites, including bones, skin, and brain; (ii) critically present their therapeutic potential against bone, skin, and neuronal diseases (e.g., osteoporosis, non-healing wounds and autoimmune skin illnesses, mood, behavior, memory, and cognitive impairments); (iii) address the current gaps in the preclinical and clinical research; and (iv) indicate new research directions and suggest future investigations.

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 upregulated 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 4 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 poorly 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 14H inhibitor. SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing 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.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain, isolated recently from feta-type cheese. Its probiotic potential has been demonstrated in a series of established in vitro tests. Moreover, incorporation of L. paracasei K5 as starter culture offered organoleptic and technological advantages to novel fermented food products. In the present study, further investigation of the potential probiotic activity of L. paracasei K5 was performed and its mechanisms of action were investigated. Employing quantitative analysis and confocal, fluorescent microscopy the adhesion properties of the above strain were studied. L. paracasei K5 displayed efficient adherence capacity to Caco-2 colon cancer cells, similarly to the reference strains Lactobacillus casei ATCC 393 and Lactobacillus rhamnosus GG. Moreover, treatment of Caco-2 cells with L. paracasei K5 inhibited cell proliferation in a time-and dose-dependent manner. The anti-proliferative effects appear to be mediated through induction of apoptosis via modulation of expression of specific Bcl-2 family proteins. These results elucidate the mechanisms of action of L. paracasei K5 and enhance its potential probiotic activity.

CD8+ T‐lymphocytes infiltration is a favorable prognostic marker in ovarian cancer. Recently we identified MEIS1 as a gene overexpressed in early stage ovarian tumors enriched for CD8+ T‐cells. Here, we report the molecular mechanism of the homeodomain transcription factor MEIS1 in lymphocyte recruitment. We validated that MEIS1 expression is a positive predictor of CD8+ T cells in early stage ovarian cancer. We showed that MEIS1 induces the expression of CCL18, CCL4, CXCL7, CCL5, CXCL1, and IL8 chemokines in cancer cells followed by their secretion in the culture medium ultimately triggering CD8+ T‐lymphocyte recruitment in vitro. Knock down of MEIS1 expression by siRNA resulted in downregulation of these chemokines. We verified that MEIS1 binds to the promoters of chemokine genes, both in vitro and in vivo. We also showed that the expression levels of MEIS1 correlated tightly with the mRNA levels of chemokines CCL4 and CCL18 in early stage ovarian cancer patient samples and served as a positive prognostic marker, as shown by Kaplan‐Meyer survival analysis. In conclusion, we propose that MEIS1 plays a pivotal role in the regulatory circuitry governing T‐cell chemo‐attraction during the early stages of ovarian cancer.

Lactobacillus pentosusB281 andLactobacillus plantarumB282 are twoLactobacillusstrains previously isolated from fermented table olives. Both strains were found to possess probiotic properties and displayed desirable technological characteristics for application as starters in novel functional food production. In the present study the anti-proliferative and immunostimulatory activities of the two strains were investigated. Firstly, we demonstrated that liveL. pentosusB281 andL. plantarumB282 significantly inhibited the growth of human colon cancer cells (Caco-2) in a time- and dose-dependent manner. By employing the air pouch system in mice, we showed that administration of both strains led to a rapid and statistically significant infiltration of leukocytes in the air pouch exudates. The phenotypical characterisation of the recruited immune cells was performed by flow cytometry analysis. We demonstrated that the majority of the infiltrated leukocytes were neutrophils. Finally by using the Mouse Cytokine Array Panel A Detection Antibody cocktail, we showed that both strains induced the expression of granulocyte-colony stimulating factor, interleukin (IL)-1α, IL-1β, IL-6, chemokine (C-X-C motif) ligand (CXCL)-1, chemokine (C-C motif) ligand (CCL)-3, CCL-4, and CXCL-2 and diminished the expression levels of soluble intercellular adhesion molecule, macrophage colony-stimulating factor and metallopeptidase inhibitor 1. Our results showed that both strains display anti-proliferative and immunostimulatory properties equal or even better in some cases than those of established and commonly used probiotic strains. These findings further support the probiotic character of the two strains.

In the present study 45 lactic acid bacteria (LAB) strains were isolated from Feta-type cheese and were screened for probiotic potential in a series of established in vitro tests, including resistance to low pH, resistance to pepsin and pancreatin and tolerance to bile salts. The strain K5, which displayed properties similar to or even better than the reference strain Lactobacillus plantarum ATCC 14917, was chosen for further analysis. Firstly, multiplex PCR analysis indicated that the novel strain belongs to the paracasei species. Secondly, the susceptibility against common antibiotics was determined to ensure a safe exploitation of the potentially probiotic strain. Additionally, the performance of L. paracasei K5 as starter in the fermentation of pomegranate juice was studied to evaluate its technological properties. Finally, a novel multiplex PCR assay, based on random amplified polymorphic DNA (RAPD) analysis was developed for its efficient and accurate detection in food products.

The beneficial effects of two potential probiotic lactobacillus strains, namely Lactobacillus pentosus B281 and L. plantarum B282 were examined and the mechanisms of action were investigated. Quantitative analysis and confocal microscopy showed that both strains exhibited a significant higher adherence to Caco-2 cells in comparison to the reference strain L. casei ATCC 393. Treatment with conditioned media (CM) of the two strains caused significant reduction of cell proliferation, as demonstrated by Sulforhodamine B (SRB) and clonogenic assays. Moreover, the CM of the two strains induced a G1-phase arrest and down-regulation of specific cyclin genes, as indicated by flow cytometry and real-time PCR analysis. To begin elucidating the nature of the bacterial components conveying these responses, the anti-proliferative effect of heat-treated CM was analysed. The anti-proliferative activity of heat-treated CM was similar to the non-heated CM in a time- and dose-dependent manner, indicating the presence of thermostable bioactive compounds.

Various natural antioxidants have been used for the development of anti-ageing cosmeceuticals due to the fact that UVR induced oxidative stress is a crucial step for the induction of photo-ageing1-4. Here we collected propolis extracts from different geographic regions of Greece and studied their anti-oxidant, anti-mutagenic and anti-ageing properties on human keratinocyte HaCat cells exposed to UVR. First we explored the cytotoxicity of propolis extracts against HaCat cells through MTT and SRB assays and estimated the safe working range of concentrations causing less than 10% loss of viability for the subsequent experiments. Next, we examined the antioxidant content of UVB irradiated HaCat cells in the presence of 20μg/ml of propolis extracts. Furthermore, we studied the antimutagenic propertises of propolis extracts by estimating DNA damage (single and double strand breaks, oxidized purines, oxidized pyrimidines and 8-oxo-2u0027-deoxyguanosine) of UVA and UVB exposed HaCat cells treated with 20μg/ml of propolis extracts. Finally, we examined the effect of propolis extracts on the UVC-induced up-regulation of MMPs at transcriptional levels. Our results have shown that propolis extracts exhibit significant anti-oxidant and anti-mutagenic properties and therefore, they are promising products for the development of anti-ageing cosmeceuticals.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.