Simultaneously Targeting Two Coupled Signalling Molecules in the Mesenchymal Stem Cell Support Efficiently Sensitises the Multiple Myeloma Cell Line H929 to Bortezomib.

Several studies have shown that diverse components of the bone marrow (BM) microenvironment play a central role in the progression, pathophysiology, and drug resistance in multiple myeloma (MM). In particular, the dynamic interaction between BM mesenchymal stem cells (BM-MSC) and MM cells has shown great relevance. Here we showed that inhibiting both PKC and NF-κB signalling pathways in BM-MSC reduced cell survival in the MM cell line H929 and increased its susceptibility to the proteasome inhibitor bortezomib. PKC-mediated cell survival inhibition and bortezomib susceptibility induction were better performed by the chimeric peptide HKPS than by the classical enzastaurin inhibitor, probably due to its greatest ability to inhibit cell adhesion and its increased capability to counteract the NF-κB-related signalling molecules increased by the co-cultivation of BM-MSC with H929 cells. Thus, inhibiting two coupled signalling molecules in BM-MSC was more effective in blocking the supportive cues emerging from the mesenchymal stroma. Considering that H929 cells were also directly susceptible to PKC and NF-κB inhibition, we showed that treatment of co-cultures with the HKPS peptide and BAY11-7082, followed by bortezomib, increased H929 cell death. Therefore, targeting simultaneously connected signalling elements of BM-MSC responsible for MM cells support with compounds that also have anti-MM activity can be an improved treatment strategy.

Efficacy of psychological interventions for young adults with mild-to-moderate depressive symptoms: A meta-analysis.

BACKGROUND: Psychological interventions are commonly used to treat mild-to-moderate depression, but their efficacy in young adults has not been exhaustively addressed. This meta-analysis aims to establish it in comparison to no treatment, wait-list, usual treatment, passive interventions, and other bona-fide treatments. METHODS: The search was conducted in Scopus, MEDLINE, PsycINFO, ClinicalTrials.gov, the ISRCTN Registry, Cochrane CENTRAL, Clarivate BIOSIS Previews and the METAPSY database, retrieving studies from the start of records to April 2020. Eligibility criteria included samples of 16-30 years experiencing mild-to-moderate depressive symptoms and participating in randomized controlled trials (RCTs), non-RCTs, or pre-post studies measuring depressive symptomatology and featuring psychological treatments. RESULTS: Up to 45 studies met criteria, consisting of 3,947 participants, assessed using the Quality Assessment Tool for Quantitative Studies and their results meta-analyzed assuming random effects. Psychological interventions proved to be efficacious in RCTs compared to no treatment (g = -0.68; 95% CI = -0.87, -0.48) and wait-list (g = -1.04; 95% CI = -1.25, -0.82), while depressive symptoms also improved in pre-post studies (g = -0.99; 95% CI = -1.32, -0.66). However, intervention efficacy was similar to usual care, passive, and bona-fide comparators. The heterogeneity found, a likely reporting bias and the low quality of most studies must be considered when interpreting these results. CONCLUSIONS: Psychological treatments are efficacious to reduce depressive symptoms in young adults, but comparable to other interventions in the mild-to-moderate range. Moderators like depression severity or therapist involvement significantly influenced their efficacy, with results encouraging clinicians to adopt flexible and personalized approaches.

Decellularization for whole organ bioengineering.

Organ transplantation in an orthotopic location is the current treatment for end-stage organ failure. However, the need for transplantable organs far exceeds the number of available donor organs. As a result, new options, such as tissue engineering and regenerative medicine, have been explored to achieve functional organ replacement. Although there have been many advances in the laboratory leading to the reconstruction of tissue and organ structures in vitro, these efforts have fallen short of producing organs that contain intact vascular networks capable of nutrient and gas exchange and are suitable for transplantation. Recently, advances in whole organ decellularization techniques have enabled the fabrication of scaffolds for engineering new organs. These scaffolds, consisting of naturally-derived extracellular matrix (ECM), provide biological signals and maintain tissue microarchitecture, including intact vascular systems that could integrate into the recipient's circulatory system. The decellularization techniques have led to the development of scaffolds for multiple organs, including the heart, liver, lung and kidney. While the experimental studies involving the use of decellularized organ scaffolds are encouraging, the translation of whole organ engineering into the clinic is still distant. This paper reviews recently described techniques used to decellularize whole organs such as the heart, lung, liver and kidney and describes possible methods for using these matrices for whole organ engineering.

Raman characterization of single-walled nanotubes of various diameters obtained by catalytic disproportionation of CO.

Single-walled carbon nanotubes prepared by disproportionation of CO over Co-Mo/SiO2 catalysts have been characterized by Raman spectroscopy, using several excitation energies. By varying the reaction temperature, different ranges of nanotube diameter were obtained. The average diameter of a single-walled nanotube produced at 750 degrees C was 0.9 nm, while it increased up to about 1.5 nm when the synthesis was conducted at 950 degrees C. The analysis of the Raman spectra obtained with a range of laser excitation energies not only gives a definite description of the single-walled nanotubes diameters but also helps differentiate the metallic or semiconducting character of the samples. This analysis can be done by comparing the experimental data with calculated gap energies as a function of nanotube diameter as well as comparing the relative intensity of bands centered at 50-60 cm-1 lower than the tangential G mode. The analysis of this feature, which can be fitted with a Breit-Wigner-Fano line, offers a method for distinguishing between metallic and semiconducting single-walled carbon nanotubes.

Mitotic phosphorylation prevents the binding of HMGN proteins to chromatin.

Condensation of the chromatin fiber and transcriptional inhibition during mitosis is associated with the redistribution of many DNA- and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quantitative gel analysis with proteins extracted from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing negative charges in the NBD demonstrate that the negative charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the negative charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1(-/-) cells indicates that the negatively charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.

Targeting of high mobility group-14/-17 proteins in chromatin is independent of DNA sequence.

Chromosomal proteins high mobility group (HMG)-14 and HMG-17 are nucleosomal-binding proteins that unfold the chromatin fiber and enhance transcription from chromatin templates. Their intracellular organization is dynamic and related to both cell cycle and transcription. Here we examine possible mechanisms for targeting HMG-14/-17 to specific regions in chromatin. Chromatin immunoprecipitation assays indicate that HMG-17 protein is not preferentially associated with chromatin regions containing transcriptionally active genes, or any type of specific DNA. We used a modification of the random amplified polymorphic DNA method to analyze DNA in various HMG-14/-17.nucleosome complexes. We found that although HMG-14 or HMG-17 proteins preferentially associate with core particles in which the DNA has a low frequency of CG dinucleotides, the genome does not contain consensus sequences that serve as specific targeting sites for the binding of either HMG-14 or HMG-17 proteins to nucleosomes. We used size exclusion and ion exchange chromatography to demonstrate that nuclei contain a large portion of HMG-17 associated with other proteins in a multiprotein complex. We suggest that these complexes regulate the dynamic organization of HMG-14/-17 in the nucleus and serve to target the proteins to specific sites in chromatin.

The accessibility of histone H3 tails in chromatin modulates their acetylation by P300/CBP-associated factor.

P300/CBP-associated factor (PCAF) is a transcriptional coactivator with intrinsic histone acetylase activity. Reversible acetylation of the core histone tails in chromatin has been linked to transcriptional regulation. Here we investigate the mechanism whereby PCAF acetylates its target in chromatin. We demonstrate that recombinant PACF preferentially acetylates the H3 tail in oligonucleosomes, as compared with nucleosome core particles. The rate of acetylation is directly related to the length of the oligonucleosomal substrate. Using a trypsin accessibility assay, we demonstrate that the rate of acetylation is also related to the accessibility of the H3 tail in uncondensed oligonucleosomes. We suggest that PCAF, and perhaps other histone acetyltransferases, acetylate chromatin templates more efficiently than core particle subunits and that this preference arises from an increased accessibility of the H3 tail in either condensed or uncondensed oligonucleosomes. Acetylation of the H3 tails by the native PCAF complex is not affected by the length of the oligonucleosomal substrate. Our results suggest that the accessibility of the H3 tail in chromatin is a major factor affecting their rate of acetylation and that component(s) in the native PCAF complex function to modify the organization of these tails in chromatin thereby enhancing their accessibility to PCAF.

Acetylation of novel sites in the nucleosomal binding domain of chromosomal protein HMG-14 by p300 alters its interaction with nucleosomes.

The reversible acetylation of histones is associated with structural alterations in the chromatin fiber that affect various DNA-related activities. Here we show that the histone acetyltransferase p300 specifically acetylates HMG-14, a nonhistone structural protein that binds to nucleosomes and reduces the compactness of the chromatin fiber. We identify 7 major acetylation sites, 6 of which are novel and have not been known to be acetylated in either HMG-14 or the closely related HMG-17 protein. All the acetylation sites involve evolutionarily conserved residues: 3 within the HMG-14/-17 nucleosomal binding domain and 4 in or near the bipartite nuclear localization domains of the proteins. In tissue culture cells the acetylation pattern is indicative of a selective process in which a subfraction of HMG-14 is preferentially acetylated. We find that the nucleosomal binding domain is a major target for acetylation in vivo and that the specific acetylation of HMG-14 by p300 weakens its interaction with nucleosome cores. Our results suggest that p300 modulates the interaction of HMG-14 with nucleosomes. Thus, p300 may affect chromatin-related activities not only by modifying histones or transcription factors but also by targeting structural nonhistone proteins.

Histone H1 is a specific repressor of core histone acetylation in chromatin.

Although a link between histone acetylation and transcription has been established, it is not clear how acetylases function in the nucleus of the cell and how they access their targets in a chromatin fiber containing H1 and folded into a highly condensed structure. Here we show that the histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF), either alone or in a nuclear complex, can readily acetylate oligonucleosomal substrates. The linker histones, H1 and H5, specifically inhibit the acetylation of mono- and oligonucleosomes and not that of free histones or histone-DNA mixtures. We demonstrate that the inhibition is due mainly to steric hindrance of H3 by the tails of linker histones and not to condensation of the chromatin fiber. Cellular PCAF, which is complexed with accessory proteins in a multiprotein complex, can overcome the linker histone repression. We suggest that linker histones hinder access of PCAF, and perhaps other HATs, to their target acetylation sites and that perturbation of the linker histone organization in chromatin is a prerequisite for efficient acetylation of the histone tails in nucleosomes.

Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes.

Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin. Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF. Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant. Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site. By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin. Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.

DNA damage activates p53 through a phosphorylation-acetylation cascade.

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

The histone acetyltransferase activity of human GCN5 and PCAF is stabilized by coenzymes.

Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases.

Clusters of nucleosomes containing chromosomal protein HMG-17 in chromatin.

Chromosomal proteins HMG-14 and HMG-17 are nucleosome binding proteins which can function as architectural elements to alter the structure of the chromatin fiber and enhance transcription from chromatin templates. Here we study the spatial organization of these HMG proteins in the nucleus and the distribution of nucleosomes containing HMG-17 in the chromatin fiber. By confocal immunofluorescence microscopy we find that HMG-14/17 proteins are clustered into foci containing either HMG-14 or HMG-17. These results suggest that HMG-14/17 proteins segregate into distinct nuclear domains. Indeed, immunofractionation of defined length oligonucleosomes, with affinity pure antibodies to HMG-17, indicates that oligonucleosomes containing HMG-17 are devoid of HMG-14. Quantitative analysis indicates that in cellular chromatin nucleosomes containing HMG-17 are clustered. The average size of the cluster is six contiguous HMG-17-containing nucleosomes. The nucleosomes in this cluster contain either two or zero molecules of HMG-17 and a complete set of four core histones. We suggest that HMG-14/17 proteins modify the nucleosomal organization of the 30 nm chromatin fiber, to unfold the higher order chromatin structure and facilitate access to the underlying DNA sequence. Clustering of architectural elements, such as HMG proteins and linker histone subtypes into distinct domains, may lead to structural and functional heterogeneity along the chromatin fiber.