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Papers by Daniela Platano

The 8th International Congress on Stress Proteins in Biology and Medicine, Stress Management Mechanisms and Pathways,CSSI 2017, 2017

☯ These authors contributed equally to this work.

Amino Acids, 2011

Polyamines are naturally occurring, positively charged polycations which are able to control seve... more Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

Studi e sperimentazioni di campo dimostrano che alcuni microorganismi appartenenti al genere Baci... more Studi e sperimentazioni di campo dimostrano che alcuni microorganismi appartenenti al genere Bacillus possono essere utilizzati efficacemente come probiotici per la sanificazione delle superfici nosocomiali, a causa della loro azione antibatterica e antifungina, essendo peraltro sicuri per la salute umana e l’ambiente. Oltre a trovare impiego in agricoltura, nella alimentazione umana e animale, nella produzione di biopolimeri e come agenti immunostimolanti per aiutare il trattamento delle malattie del tratto gastrointestinale e urinario, in generale i probiotici sono in grado di contrastare efficacemente e nel tempo la proliferazione di altre specie batteriche potenzialmente patogene. A sostegno della sicurezza nell’impiego dei Bacillus, vengono inoltre descritte alcuni tipi di indagini (antibiogrammi e analisi molecolari) che, oltre ad constatare l’efficacia, nei confronti di queste specie microbiche, della maggior parte degli antibiotici, in grado di verificare e monitorare il livello di sicurezza dei prodotti probiotici usati

Proceedings of the National Academy of Sciences, 1998

β subunits of voltage-gated Ca 2+ channels are encoded in four genes and display additional molec... more β subunits of voltage-gated Ca 2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for β2a, which differs in that it does not support prepulse facilitation of α 1C Ca 2+ channels, inhibits voltage-induced inactivation of neuronal α 1E Ca 2+ channels, and is more effective in blocking inhibition of α 1E channels by G protein-coupled receptors. We show that the distinguishing properties of β2a, rather than interaction with a distinct site of α 1 , are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of β2a were lost in a mutant that could not be palmitoylated [β2a(Cys 3,4 Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-depende...

Free Radical Biology and Medicine

J Bioenerg Biomembrane, 1998

Background: The non-canonical NF-kB activating kinase IKKa, encoded by CHUK (conserved-helix-loop... more Background: The non-canonical NF-kB activating kinase IKKa, encoded by CHUK (conserved-helix-loop-helix-ubiquitouskinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKa as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKKa expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKaf/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKa in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKa ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKa-deficient chondrocytes. IKKa deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKa-deficient chondrocytes was rescued by a kinase-dead IKKa protein mutant. Conclusions/Significance: IKKa acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKa positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Tgus, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

PLOS ONE, 2015

Introduction Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (... more Introduction Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. Methods In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.

PLoS ONE, 2014

Background: Healthcare-Associated Infections (HAIs) are one of the most frequent complications oc... more Background: Healthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcareassociated pathogens, thus indicating the need for new and sustainable strategies. Aim: This study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting. Methods: The effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected. Results: Microbial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3-4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m 2 when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities. Conclusions: This study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAIrelated microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.

Journal of Neuroscience, 2013

A newly formed memory is temporarily fragile and becomes stable through a process known as consol... more A newly formed memory is temporarily fragile and becomes stable through a process known as consolidation. Stable memories may again become fragile if retrieved or reactivated, and undergo a process of reconsolidation to persist and strengthen. Both consolidation and reconsolidation require an initial phase of transcription and translation that lasts for several hours. The identification of the critical players of this gene expression is key for understanding long-term memory formation and persistence. In rats, the consolidation of inhibitory avoidance (IA) memory requires gene expression in both the hippocampus and amygdala, two brain regions that process contextual/spatial and emotional information, respectively; IA reconsolidation requires de novo gene expression in the amygdala. Here we report that, after IA learning, the levels of the transcription factor CCAAT enhancer binding protein ␦ (C/EBP␦) are significantly increased in both the hippocampus and amygdala. These increases are essential for long-term memory consolidation, as their blockade via antisense oligodeoxynucleotide-mediated knockdown leads to memory impairment. Furthermore, C/EBP␦ is upregulated and required in the amygdala for IA memory reconsolidation. C/EBP␦ is found in nuclear, somatic, and dendritic compartments, and a dendritic localization of C/EBP␦ mRNA in hippocampal neuronal cultures suggests that this transcription factor may be translated at synapses. Finally, the induction of long-term potentiation at CA3-CA1 synapses by tetanic stimuli in acute slices, a cellular model of long-term memory, leads to an accumulation of C/EBP␦ in the nucleus. We conclude that the transcription factor C/EBP␦ plays a critical role in memory consolidation and reconsolidation.

Arthritis Research & Therapy, 2014

Introduction: Recent evidence suggests that tissue accumulation of senescent p16 INK4a-positive c... more Introduction: Recent evidence suggests that tissue accumulation of senescent p16 INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16 INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16 INK4a in the OA-induced SASP and its regulation by microRNAs (miRs). Methods: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16 INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16 INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis. Results: p16 INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16 INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16 INK4a. Accordingly, p16 INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis. Conclusions: We disclosed herein a new role of the senescence marker p16 INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Rejuvenation Research, 2012

Aging is associated with a gradual decline in cognitive functions, and more dramatic cognitive im... more Aging is associated with a gradual decline in cognitive functions, and more dramatic cognitive impairments occur in patients affected by Alzheimer's disease (AD). Electrophysiological and molecular studies performed in aged animals and in animal models of AD have shown that cognitive decline is associated with significant modifications in synaptic plasticity (i.e., activity-dependent changes in synaptic strength) and have elucidated some of the cellular mechanisms underlying this process. Morphological studies have revealed a correlation between the quality of memory performance and the extent of structural changes of synaptic contacts occurring during memory consolidation. We briefly review recent experimental evidence here.

Rejuvenation Research, 2010

Microarray analysis was used to identify genes differentially expressed in the hippocampus of age... more Microarray analysis was used to identify genes differentially expressed in the hippocampus of aged rats showing diverse long-term (3 and 6 h) spatial-associative memory performance in a single-trial inhibitory avoidance task. The transcription of 43 genes (including genes functionally linked to signal transduction, cell growth and differentiation, translation, energy metabolism, and nucleic acid processing) was significantly upregulated in good- versus bad-performing animals, whereas that of 18 genes (including genes functionally linked to transcription, cell growth and differentiation, apoptosis, and protein transport) was significantly downregulated in good- versus bad-performing animals. The differential expression of 14 of these genes was confirmed by real-time polymerase chain reaction.

Rejuvenation Research, 2008

Aging is associated with deficits in long-term declarative memory formation, and wide differences... more Aging is associated with deficits in long-term declarative memory formation, and wide differences in performance can be observed among aged individuals. The cellular substrates of these deficits and the reasons for such marked individual differences are not yet fully understood. In the present study, morphologic parameters of synapses and synaptic mitochondria in stratum molecolare of CA1 hippocampal region were investigated in aged (26- to 27-month-old) female rats after a single trial inhibitory avoidance task. In this memory protocol animals learn to avoid a dark compartment in which they received a mild, inescapable foot shock. Rats were tested 3 and 6 or 9 hours after the training, divided into good and bad responders according to their performance (retention times above or below 100 seconds, respectively) and immediately sacrificed. The number of synapses and synaptic mitochondria per cubic micrometer of tissue (numeric density), the average area of synapses and volume of synaptic mitochondria, the total area of synapses per cubic micrometer of tissue, the percentage of perforated synapses and the overall volume of mitochondria per cubic micrometer of tissue were evaluated. In the good responder group, the numeric density of synapses and mitochondria was significantly higher and the average mitochondrial volume was significantly smaller 9 hours versus 6 hours after the training. No significant differences were observed among bad responders. Thus, better performances in passive avoidance memory task are correlated with more efficient plastic remodeling of synaptic contacts and mitochondria in hippocampal CA1. Present findings indicate that maintenance of synaptic plastic reactivity during aging is a critical requirement for preserving long-term memory consolidation.

Rejuvenation Research, 2006

The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-mont... more The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-month-old) and old (25- to 27-month-old) rats exposed to a six-session object recognition visual training were investigated by morphometric methods. The comparative analysis showed a higher synaptic numeric density, a lower synaptic average area, and a lower percentage of megasynapses (S > 0.5 microm2) in old trained rats versus controls, and a higher percentage of small (S < 0.15 microm2) junctions in adult trained rats versus controls. The more marked synaptic remodeling underlying memory consolidation in the perirhinal cortex of old rats might reflect a pre-existing lower dynamic status.

Rejuvenation Research, 2008

Aging is associated with memory impairments, but the neural bases of this process need to be clar... more Aging is associated with memory impairments, but the neural bases of this process need to be clarified. To this end, behavioral protocols for memory testing may be applied to aged animals to compare memory performances with functional and structural characteristics of specific brain regions. Visual object recognition memory can be investigated in the rat using a behavioral task based on its spontaneous preference for exploring novel rather than familiar objects. We found that a behavioral task able to elicit long-term visual object recognition memory in adult Long-Evans rats failed in aged (25-27 months old) Wistar rats. Since no tasks effective in aged rats are reported in the literature, we changed the experimental conditions to improve consolidation processes to assess whether this form of memory can still be maintained for long term at this age: the learning trials were performed in a smaller box, identical to the home cage, and the inter-trial delays were shortened. We observed a reduction in anxiety in this box (as indicated by the lower number of fecal boli produced during habituation), and we developed a learning protocol able to elicit a visual object recognition memory that was maintained after 24 h in these aged rats. When we applied the same protocol to adult rats, we obtained similar results. This experimental approach can be useful to study functional and structural changes associated with age-related memory impairments, and may help to identify new behavioral strategies and molecular targets that can be addressed to ameliorate memory performances during aging.

Proceedings of the National Academy of Sciences, 1997

Several classes of voltage-gated Ca 2؉ channels (VGCCs) are inhibited by G proteins activated by ... more Several classes of voltage-gated Ca 2؉ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca 2؉ channels the executing arm of the activated G protein is its ␤␥ dimer (G␤␥). We report below the existence of two G␤␥-binding sites on the A-, Band nd E-type ␣ 1 subunits that form non-L-type Ca 2؉ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both G␤␥-binding regions also bind the Ca 2؉ channel ␤ subunit (CC␤), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in ␣ 1E of loop 1 with that of the G proteininsensitive and G␤␥-binding-negative loop 1 of ␣ 1C did not abolish inhibition by G proteins, but the exchange of the ␣ 1E C terminus with that of ␣ 1C did. This and properties of ␣ 1E C-terminal truncations indicated that the G␤␥-binding site mediating the inhibition of Ca 2؉ channel activity is the one in the C terminus. Binding of G␤␥ to this site was inhibited by an ␣ 1-binding domain of CC␤, thus providing an explanation for the functional antagonism existing between CC␤ and G protein inhibition. The data do not support proposals that G␤␥ inhibits ␣ 1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Proceedings of the National Academy of Sciences, 1996

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of c... more During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2؉ homeostasis and the physiological roles of CCE.

The 8th International Congress on Stress Proteins in Biology and Medicine, Stress Management Mechanisms and Pathways,CSSI 2017, 2017

☯ These authors contributed equally to this work.

Amino Acids, 2011

Polyamines are naturally occurring, positively charged polycations which are able to control seve... more Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

Studi e sperimentazioni di campo dimostrano che alcuni microorganismi appartenenti al genere Baci... more Studi e sperimentazioni di campo dimostrano che alcuni microorganismi appartenenti al genere Bacillus possono essere utilizzati efficacemente come probiotici per la sanificazione delle superfici nosocomiali, a causa della loro azione antibatterica e antifungina, essendo peraltro sicuri per la salute umana e l’ambiente. Oltre a trovare impiego in agricoltura, nella alimentazione umana e animale, nella produzione di biopolimeri e come agenti immunostimolanti per aiutare il trattamento delle malattie del tratto gastrointestinale e urinario, in generale i probiotici sono in grado di contrastare efficacemente e nel tempo la proliferazione di altre specie batteriche potenzialmente patogene. A sostegno della sicurezza nell’impiego dei Bacillus, vengono inoltre descritte alcuni tipi di indagini (antibiogrammi e analisi molecolari) che, oltre ad constatare l’efficacia, nei confronti di queste specie microbiche, della maggior parte degli antibiotici, in grado di verificare e monitorare il livello di sicurezza dei prodotti probiotici usati

Proceedings of the National Academy of Sciences, 1998

β subunits of voltage-gated Ca 2+ channels are encoded in four genes and display additional molec... more β subunits of voltage-gated Ca 2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for β2a, which differs in that it does not support prepulse facilitation of α 1C Ca 2+ channels, inhibits voltage-induced inactivation of neuronal α 1E Ca 2+ channels, and is more effective in blocking inhibition of α 1E channels by G protein-coupled receptors. We show that the distinguishing properties of β2a, rather than interaction with a distinct site of α 1 , are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of β2a were lost in a mutant that could not be palmitoylated [β2a(Cys 3,4 Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-depende...

Free Radical Biology and Medicine

J Bioenerg Biomembrane, 1998

Background: The non-canonical NF-kB activating kinase IKKa, encoded by CHUK (conserved-helix-loop... more Background: The non-canonical NF-kB activating kinase IKKa, encoded by CHUK (conserved-helix-loop-helix-ubiquitouskinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKa as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKKa expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKaf/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKa in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKa ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKa-deficient chondrocytes. IKKa deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKa-deficient chondrocytes was rescued by a kinase-dead IKKa protein mutant. Conclusions/Significance: IKKa acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKa positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Tgus, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

PLOS ONE, 2015

Introduction Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (... more Introduction Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. Methods In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.

PLoS ONE, 2014

Background: Healthcare-Associated Infections (HAIs) are one of the most frequent complications oc... more Background: Healthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcareassociated pathogens, thus indicating the need for new and sustainable strategies. Aim: This study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting. Methods: The effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected. Results: Microbial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3-4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m 2 when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities. Conclusions: This study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAIrelated microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.

Journal of Neuroscience, 2013

A newly formed memory is temporarily fragile and becomes stable through a process known as consol... more A newly formed memory is temporarily fragile and becomes stable through a process known as consolidation. Stable memories may again become fragile if retrieved or reactivated, and undergo a process of reconsolidation to persist and strengthen. Both consolidation and reconsolidation require an initial phase of transcription and translation that lasts for several hours. The identification of the critical players of this gene expression is key for understanding long-term memory formation and persistence. In rats, the consolidation of inhibitory avoidance (IA) memory requires gene expression in both the hippocampus and amygdala, two brain regions that process contextual/spatial and emotional information, respectively; IA reconsolidation requires de novo gene expression in the amygdala. Here we report that, after IA learning, the levels of the transcription factor CCAAT enhancer binding protein ␦ (C/EBP␦) are significantly increased in both the hippocampus and amygdala. These increases are essential for long-term memory consolidation, as their blockade via antisense oligodeoxynucleotide-mediated knockdown leads to memory impairment. Furthermore, C/EBP␦ is upregulated and required in the amygdala for IA memory reconsolidation. C/EBP␦ is found in nuclear, somatic, and dendritic compartments, and a dendritic localization of C/EBP␦ mRNA in hippocampal neuronal cultures suggests that this transcription factor may be translated at synapses. Finally, the induction of long-term potentiation at CA3-CA1 synapses by tetanic stimuli in acute slices, a cellular model of long-term memory, leads to an accumulation of C/EBP␦ in the nucleus. We conclude that the transcription factor C/EBP␦ plays a critical role in memory consolidation and reconsolidation.

Arthritis Research & Therapy, 2014

Introduction: Recent evidence suggests that tissue accumulation of senescent p16 INK4a-positive c... more Introduction: Recent evidence suggests that tissue accumulation of senescent p16 INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16 INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16 INK4a in the OA-induced SASP and its regulation by microRNAs (miRs). Methods: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16 INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16 INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis. Results: p16 INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16 INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16 INK4a. Accordingly, p16 INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis. Conclusions: We disclosed herein a new role of the senescence marker p16 INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Rejuvenation Research, 2012

Aging is associated with a gradual decline in cognitive functions, and more dramatic cognitive im... more Aging is associated with a gradual decline in cognitive functions, and more dramatic cognitive impairments occur in patients affected by Alzheimer's disease (AD). Electrophysiological and molecular studies performed in aged animals and in animal models of AD have shown that cognitive decline is associated with significant modifications in synaptic plasticity (i.e., activity-dependent changes in synaptic strength) and have elucidated some of the cellular mechanisms underlying this process. Morphological studies have revealed a correlation between the quality of memory performance and the extent of structural changes of synaptic contacts occurring during memory consolidation. We briefly review recent experimental evidence here.

Rejuvenation Research, 2010

Microarray analysis was used to identify genes differentially expressed in the hippocampus of age... more Microarray analysis was used to identify genes differentially expressed in the hippocampus of aged rats showing diverse long-term (3 and 6 h) spatial-associative memory performance in a single-trial inhibitory avoidance task. The transcription of 43 genes (including genes functionally linked to signal transduction, cell growth and differentiation, translation, energy metabolism, and nucleic acid processing) was significantly upregulated in good- versus bad-performing animals, whereas that of 18 genes (including genes functionally linked to transcription, cell growth and differentiation, apoptosis, and protein transport) was significantly downregulated in good- versus bad-performing animals. The differential expression of 14 of these genes was confirmed by real-time polymerase chain reaction.

Rejuvenation Research, 2008

Aging is associated with deficits in long-term declarative memory formation, and wide differences... more Aging is associated with deficits in long-term declarative memory formation, and wide differences in performance can be observed among aged individuals. The cellular substrates of these deficits and the reasons for such marked individual differences are not yet fully understood. In the present study, morphologic parameters of synapses and synaptic mitochondria in stratum molecolare of CA1 hippocampal region were investigated in aged (26- to 27-month-old) female rats after a single trial inhibitory avoidance task. In this memory protocol animals learn to avoid a dark compartment in which they received a mild, inescapable foot shock. Rats were tested 3 and 6 or 9 hours after the training, divided into good and bad responders according to their performance (retention times above or below 100 seconds, respectively) and immediately sacrificed. The number of synapses and synaptic mitochondria per cubic micrometer of tissue (numeric density), the average area of synapses and volume of synaptic mitochondria, the total area of synapses per cubic micrometer of tissue, the percentage of perforated synapses and the overall volume of mitochondria per cubic micrometer of tissue were evaluated. In the good responder group, the numeric density of synapses and mitochondria was significantly higher and the average mitochondrial volume was significantly smaller 9 hours versus 6 hours after the training. No significant differences were observed among bad responders. Thus, better performances in passive avoidance memory task are correlated with more efficient plastic remodeling of synaptic contacts and mitochondria in hippocampal CA1. Present findings indicate that maintenance of synaptic plastic reactivity during aging is a critical requirement for preserving long-term memory consolidation.

Rejuvenation Research, 2006

The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-mont... more The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-month-old) and old (25- to 27-month-old) rats exposed to a six-session object recognition visual training were investigated by morphometric methods. The comparative analysis showed a higher synaptic numeric density, a lower synaptic average area, and a lower percentage of megasynapses (S > 0.5 microm2) in old trained rats versus controls, and a higher percentage of small (S < 0.15 microm2) junctions in adult trained rats versus controls. The more marked synaptic remodeling underlying memory consolidation in the perirhinal cortex of old rats might reflect a pre-existing lower dynamic status.

Rejuvenation Research, 2008

Aging is associated with memory impairments, but the neural bases of this process need to be clar... more Aging is associated with memory impairments, but the neural bases of this process need to be clarified. To this end, behavioral protocols for memory testing may be applied to aged animals to compare memory performances with functional and structural characteristics of specific brain regions. Visual object recognition memory can be investigated in the rat using a behavioral task based on its spontaneous preference for exploring novel rather than familiar objects. We found that a behavioral task able to elicit long-term visual object recognition memory in adult Long-Evans rats failed in aged (25-27 months old) Wistar rats. Since no tasks effective in aged rats are reported in the literature, we changed the experimental conditions to improve consolidation processes to assess whether this form of memory can still be maintained for long term at this age: the learning trials were performed in a smaller box, identical to the home cage, and the inter-trial delays were shortened. We observed a reduction in anxiety in this box (as indicated by the lower number of fecal boli produced during habituation), and we developed a learning protocol able to elicit a visual object recognition memory that was maintained after 24 h in these aged rats. When we applied the same protocol to adult rats, we obtained similar results. This experimental approach can be useful to study functional and structural changes associated with age-related memory impairments, and may help to identify new behavioral strategies and molecular targets that can be addressed to ameliorate memory performances during aging.

Proceedings of the National Academy of Sciences, 1997

Several classes of voltage-gated Ca 2؉ channels (VGCCs) are inhibited by G proteins activated by ... more Several classes of voltage-gated Ca 2؉ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca 2؉ channels the executing arm of the activated G protein is its ␤␥ dimer (G␤␥). We report below the existence of two G␤␥-binding sites on the A-, Band nd E-type ␣ 1 subunits that form non-L-type Ca 2؉ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both G␤␥-binding regions also bind the Ca 2؉ channel ␤ subunit (CC␤), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in ␣ 1E of loop 1 with that of the G proteininsensitive and G␤␥-binding-negative loop 1 of ␣ 1C did not abolish inhibition by G proteins, but the exchange of the ␣ 1E C terminus with that of ␣ 1C did. This and properties of ␣ 1E C-terminal truncations indicated that the G␤␥-binding site mediating the inhibition of Ca 2؉ channel activity is the one in the C terminus. Binding of G␤␥ to this site was inhibited by an ␣ 1-binding domain of CC␤, thus providing an explanation for the functional antagonism existing between CC␤ and G protein inhibition. The data do not support proposals that G␤␥ inhibits ␣ 1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Proceedings of the National Academy of Sciences, 1996

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of c... more During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2؉ homeostasis and the physiological roles of CCE.