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.

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.

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.

Characterization of preferred deoxyribonuclease I cleavage sites.

The preferred DNase I cleavage sites within the 160 bp tyrT DNA fragment were identified by studying the initial rate of cleavage of individual bonds. The results show that there is no correlation between the rate of cleavage and the identity of the dinucleotide sequence that is cleaved. Examination of the sequences surrounding the seven most rapidly cleaved bonds suggests that an A-T base-pair is preferred at the position three bases to the 5' side of the cleavage site. Preferential cleavage at such sites is consistent with predictions based on the recently obtained high resolution structure of a DNase I-octanucleotide complex. A statistical analysis of 54 additional preferred DNase I cleavage sites, using sequence data taken from published literature, confirms that DNase I exhibits a local sequence preference in addition to its relatively well characterized global structural specificity. Our analysis indicates preferential cleavage at the sequences 5'ATYAT--ATVN, where -- indicates the cleavage site, the notation AT indicates a preference for an A-T base-pair, and V indicates not-T. Comparative kinetic studies of the digestion of three deoxyoctanucleotides by DNase I quantitatively support the sequence preference inferred from the sequence analysis. Poor DNase I cleavage sites were also examined, and found to be characterized by the sequence motif 5'GCRR--TTY. Notably, poor cleavage sites characteristically contain G or C at position -3. While DNase I certainly does not cleave with an absolute sequence specificity, our studies reveal a distinct sequence preference in DNase I cleavage that has heretofore been unappreciated and uncharacterized.

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.

Pharmacokinetics of oral nifedipine: relevance of the distribution phase.

Pharmacokinetics of oral Nifedipine was studied in 12 Mexican young healthy volunteers, six men and six women, who received a 10 mg capsule. Plasma levels were determined by a nifedipine specific HPLC assay. Experimental data were fitted and pharmacokinetic parameters were calculated using an open two compartment model. No statistically significant difference was detected between men and women, thus both sexes were considered as a single population. Nifedipine plasma levels rose rapidly (ka = 8.46 +/- 1.96 h-1) reaching a maximum concentration of 145 +/- 23 ng/ml in 0.61 +/- 0.07 h. Plasma levels then decayed with a distribution phase (alpha = 1.98 +/- 0.40 h-1, t1/2 alpha = 0.46 +/- 0.06 h) and a terminal elimination phase (beta = 0.17 +/- 0.03 h-1, t1/2 beta = 4.98 +/- 0.55 h). AUC was 384 +/- 41 ng h/ml. Values of AUC and t1/2 beta were higher than those reported by other authors. Differences in the AUC could be due to ethnic origin, environmental factors or nutritional habits. Ten subjects presented plasma concentration-time curves in which the distribution phase was clearly distinguishable, having a ka/alpha relationship higher than 1.5. For the other two subjects, the distribution phase was not apparent and ka/alpha was lower than 1.5. The results show that an adequate characterization of the distribution phase is required if one pretends to use pharmacokinetic data for dosage regimen design.

Pharmacokinetics of nifedipine slow release tablet in Mexican subjects: further evidence for an oxidation polymorphism.

Nifedipine kinetics after ingestion of 20 mg slow release tablets were studied in 12 young, healthy, Mexican subjects. Plasma levels were determined by a nifedipine-specific HPLC assay. Levels rose after drug administration reaching a maximum concentration of 48.7 +/- 7.3 ng/ml in 2.1 +/- 0.7 h (mean +/- SEM). Concentrations then decayed with a terminal half-life of 16.9 +/- 3.1 hours. AUC was 526 +/- 62 ng h/ml. Five individuals were fast and seven were slow nifedipine metabolizers, according to the AUC criterion proposed by Kleinbloesem and coworkers. Individual AUC/Dose values from this and from other two studies on oral nifedipine kinetics in Mexicans were cumulated and the frequency histogram and probit analyses were performed (N = 30). A bimodal distribution was clearly observed. Fast and slow metabolizers were distinguished as those subjects with AUC/Dose values either lower or higher than 22.5 ng h/ml mg. Unlike European populations, it appears that slow metabolization is more frequent in Mexicans. Data strongly support the hypothesis of the existence of a polymorphism concerning nifedipine disposition kinetics due to genetic basis.

Determination of oral rifampin pharmacokinetic parameters in Mexicans and comparison with other populations: absence of evidence for interethnic variability.

Oral pharmacokinetics of rifampin were studied in eight Mexican young healthy male volunteers after administration of a 600 mg oral dose. After an overnight fast, subjects received medication and blood samples were drawn at selected times over a 24-h period. Rifampin plasma levels were determined by HPLC. Pharmacokinetic parameters (mean +/- SEM) were: Cmax 13.514 +/- 1.775 micrograms/ml, tmax 1.88 +/- 0.30 h, AUC 73.61 +/- 9.48 micrograms.h/ml and half-life 2.98 +/- 0.29 h. Results were compared with those obtained for other populations under similar conditions in order to explore the possibility of interethnic variability, since it has been reported that rifampin pharmacokinetics in Indonesian subjects differ from those found in Europeans. Pharmacokinetic data found in Mexicans were comparable with those observed in British, Indian, Japanese and Italian individuals. As the pharmacokinetics of rifampin seem to be similar in different populations, it is concluded that ethnic origin does not appear to play an important role. Therefore, dosing regimens designed for Caucasians can be extrapolated for other populations.

Pharmacokinetics of oral ranitidine in Mexicans.

The pharmacokinetics of oral ranitidine were studied in 24 Mexican male healthy volunteers. Subjects received a tablet containing 150 mg of ranitidine (Azantac, Glaxo de México, Mexico City) after an overnight fast and blood samples were drawn at several times for a period of 24 h. Ranitidine concentration in plasma was measured by high performance liquid chromatography and pharmacokinetic parameters were determined by non-compartmental analysis. Ranitidine plasma concentration increased with time, reaching a maximum of (mean +/- SEM) 484 +/- 34 ng/ml in 2.7 +/- 0.2 h. Plasma levels then decayed with a terminal half-life of 4.8 +/- 0.3 h. The area under the plasma concentration against time curve was 2440 +/- 126 ngh/ml. Oral ranitidine pharmacokinetic parameters in Mexicans appeared to be similar to those previously reported for Caucasians.

Determination of Three Acetylator Phenotypes in a Mexican Population Using Sulfamethazine Metabolic Ratio.

Eighty-six Mexican young healthy volunteers received a 500-mg oral sulfamethazine dose. Urine was collected 0--6 h after medication and acetylated, and unchanged sulfamethazine were determined by the Bratton--Marshall method. The frequency distribution of acetylated/unchanged sulfamethazine, determined by probit analysis, appeared to be trimodal, allowing the discrimination of three acetylator phenotypes. The frequencies of rapid, intermediate, and slow acetylators were 28%, 42%, and 30% respectively, being consistent with the Hardy-Weinberg law.

Lymphocyte proliferation kinetics and sister-chromatid exchanges in individuals treated with metronidazole.

Metronidazole, an effective agent for the treatment of protozoan infections, is frequently used in developing countries. However, the employment of this drug has been questioned in view of its mutagenicity in bacteria and carcinogenicity in mice. A genotoxic study was carried out in which cellular proliferation kinetics and the frequency of sister-chromatid exchanges were determined in human peripheral blood lymphocytes from 12 individuals treated with therapeutic doses of metronidazole. No effect was observed on mitotic index with the treatment, although a significant increase was found in three individuals after treatment. No increase of sister-chromatid exchanges was detected. The rate of lymphocyte proliferation kinetics showed an increase after the metronidazole treatment in all patients, indicating a possible immunostimulatory action.

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.

Differences in nifedipine concentration-effect relationship between capsule and slow release tablet administration.

The relationships between nifedipine plasma concentrations and its hypotensive and positive chronotropic effects were studied in healthy volunteers who received either a 10 mg capsule (CAP) or a 20 mg slow release tablet (SRT). Plasma concentrations rose more rapidly after CAP than after SRT, Cmax being 131 +/- 39 and 40 +/- 7 ng/ml and tmax being 0.5 +/- 0.07 and 1.8 +/- 0.4 h, respectively. Both formulations produced a reduction in diastolic blood pressure which exhibited a significant linear correlation (p < 0.01) with nifedipine plasma concentration. However, the slope obtained with SRT was significantly higher than that of CAP (0.24 +/- 0.05 vs 0.07 +/- 0.01, p < 0.01). That is, a similar hypotensive effect was produced at a lower concentration with SRT than with CAP. A positive chronotropic effect which exhibited a highly significant correlation with nifedipine plasma concentration (p < 0.0001) was observed with CAP. Conversely, with SRT heart rate increase was smaller and there was no significant correlation with nifedipine plasma concentration (p > 0.45). Since the measured decrease in blood pressure is the outcome of nifedipine-induced vasodilation and of homeostatic responses, results are interpreted as follows. Fast nifedipine input after CAP induced a brisk change in physiological conditions and hence triggered an important homeostatic response, visualized as heart rate increase, which partially offset the hypotensive effect. With SRT, there was a gradual change in blood pressure producing lesser activation of compensatory mechanisms and therefore the hypotensive effect of nifedipine was less antagonized than with CAP. Nifedipine SRT does not only exhibit pharmacokinetic advantages, but also a more favorable pharmacodynamic profile than CAP.

[Agricultural products handling: methods of feasibility evaluation]. / Procesamiento artesanal de productos agrícolas: métodos de evaluación de factibilidad.

Post-harvest problems are important constraints to the expansion of production of food in many Latin American countries. Besides problems of bulkiness, perishability and seasonal production patterns, the necessity of reducing transportation costs, increasing rural employment, and finding new markets for processed products, requires the development of processing technologies. Possible processed products include a vast range of alternatives. Given limited time and resources, it is not always feasible to carry out detailed studies. Hence a practical, low-cost methodology is needed to evaluate the available options. This paper presents a series of methods to evaluate different processing possibilities. It describes in detail each method including a rapid initial assessment, market and consumer research, farm-oriented research, costs and returns analysis and finally, some marketing and promotion strategies.

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.

A premelting conformational transition in poly(dA)-Poly(dT) coupled to daunomycin binding.

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.

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.