Transcriptomic Analysis of Peripheral Monocytes upon Fingolimod Treatment in Relapsing Remitting Multiple Sclerosis Patients.

Fingolimod (FTY), a second-line oral drug approved for relapsing remitting Multiple Sclerosis (RRMS) acts in preventing lymphocyte migration outside lymph nodes; moreover, several lines of evidence suggest that it also inhibits myeloid cell activation. In this study, we investigated the transcriptional changes induced by FTY in monocytes in order to better elucidate its mechanism of action. CD14+ monocytes were collected from 24 RRMS patients sampled at baseline and after 6 months of treatment and RNA profiles were obtained through next-generation sequencing. We conducted pathway and sub-paths analysis, followed by centrality analysis of cell-specific interactomes on differentially expressed genes (DEGs). We investigated also the predictive role of baseline monocyte transcription profile in influencing the response to FTY therapy. We observed a marked down-regulation effect (60 down-regulated vs. 0 up-regulated genes). Most of the down-regulated DEGs resulted related with monocyte activation and migration like IL7R, CCR7 and the Wnt signaling mediators LEF1 and TCF7. The involvement of Wnt signaling was also confirmed by subpaths analyses. Furthermore, pathway and network analyses showed an involvement of processes related to immune function and cell migration. Baseline transcriptional profile of the HLA class II gene HLA-DQA1 and HLA-DPA1 were associated with evidence of disease activity after 2 years of treatment. Our data support the evidence that FTY induces major transcriptional changes in monocytes, mainly regarding genes involved in cell trafficking and immune cell activation. The baseline transcriptional levels of genes associated with antigen presenting function were associated with disease activity after 2 years of FTY treatment.

Activation of the SDF1/CXCR4 pathway retards muscle atrophy during cancer cachexia.

Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and causes severe body weight loss, with rapid depletion of skeletal muscle. No treatment is available. We analyzed microarray data sets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents but not in those with diabetes, disuse, uremia or fasting. Ingenuity Pathways Analysis indicated that three genes belonging to the C-X-C motif chemokine receptor 4 (CXCR4) pathway were downregulated only in muscles atrophying because of cancer: stromal cell-derived factor 1 (SDF1), adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Notably, we found that, in the Rectus Abdominis muscle of cancer patients, the expression of SDF1 and CXCR4 was inversely correlated with that of two ubiquitin ligases induced in muscle wasting, atrogin-1 and MuRF1, suggesting a possible clinical relevance of this pathway. The expression of all main SDF1 isoforms (α, ß, γ) also declined in Tibialis Anterior muscle from cachectic mice bearing murine colon adenocarcinoma or human renal cancer and drugs with anticachexia properties restored their expression. Overexpressing genes of this pathway (that is, SDF1 or CXCR4) in cachectic muscles increased the fiber area by 20%, protecting them from wasting. Similarly, atrophying myotubes treated with either SDF1α or SDF1ß had greater total protein content, resulting from reduced degradation of overall long-lived proteins. However, inhibiting CXCR4 signaling with the antagonist AMD3100 did not affect protein homeostasis in atrophying myotubes, whereas normal myotubes treated with AMD3100 showed time- and dose-dependent reductions in diameter, until a plateau, and lower total protein content. This further confirms the involvement of a saturable pathway (that is, CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer.

T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse.

Abnormal connective tissue proliferation following muscle degeneration is a major pathological feature of Duchenne muscular dystrophy (DMD), a genetic myopathy due to lack of the sarcolemmal dystrophin protein. Since this fibrotic proliferation is likely to be a major obstacle to the efficacy of future therapies, research is needed to understand and prevent the fibrotic process in order to develop an effective treatment. Murine muscular dystrophy (mdx) is genetically homologous to DMD, and histopatological alterations are comparable to those of the muscles of patients with DMD. To investigate the development of fibrosis, we bred the mdx mouse with the scid immunodepressed mouse and analysed fibrosis histologically; we used ELISA analysis to determine TGF-beta1 expression. Significant reduction of fibrosis and TGF-beta1 expression was found in the muscles of the scid/mdx mice. However, we observed similar centrally located nuclei, necrosis, muscle degeneration and muscle force compared to the mdx animals. These data demonstrate a correlation between the absence of B and T lymphocytes and loss of fibrosis accompanied by reduction of TGF-beta1, suggesting the importance of modulation of the immune system in DMD.

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil.

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation.

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.

Two-dimensional electrophoresis of Cereus peruvianus (Cactaceae) callus tissue proteins.

Two-dimensional electrophoresis of Cereus peruvianus callus tissues grown in culture media containing two different 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin combinations was used to identify minor differences in polypeptide composition of these cell clones. Altered expression during growth in the two 2,4-D and kinetin combinations was apparent for 13 polypeptides when calluses in the two media were compared. The number of proteins with differential expression (presence or absence of specific spots) was higher in callus tissues cultured in the 4.0 mg/L 2,4-D and 8.0 mg/L kinetin combination than in callus tissues cultured in the 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination. The present results show that the callus tissues maintained at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin can be used as a matrix for in vitro selection programs.

Identification of a DNA-binding factor that recognizes an alpha-coixin promoter and interacts with a Coix Opaque-2 like protein.

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the alpha-coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from -1084 to -238. However, deletion from -238 to -158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The -238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same retarded complexes were observed when the -158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the alpha-coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the -238/ATG promoter fragment showed that a 35 bp region from -86 to -51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line.

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0-93 ⋅ 10-6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10-6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10-6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1.

Ventricular pacing threshold and refractoriness after defibrillation shocks in patients with implantable cardioverter-defibrillators.

The aim of this study was to examine the effect of ventricular fibrillation and a subsequent defibrillation shock on ventricular excitability and refractoriness in human beings. We studied 16 consecutive patients with implantable cardioverter-defibrillators undergoing follow-up studies. The pre- and post-shock pacing threshold, ventricular effective refractory period, monophasic action potential duration, and serum catecholamine levels were measured. Compared with the baseline state, immediately after ventricular fibrillation, and a successful defibrillation shock: (1) the ventricular effective refractory period decreased from 251 +/- 24 ms to 222 +/- 30 ms (p < 0.01), (2) the monophasic action potential duration decreased from 210 +/- 16 ms to 179 +/- 23 ms (P < 0.01) at 50% repolarization and from 274 +/- 24 ms to 240 +/- 26 ms (P< 0.01) at 90% repolarization, (3) the pacing threshold was not significantly altered and, (4) serum levels of epinephrine and norepinephrine were elevated. These results show that although ventricular fibrillation and subsequent defibrillation had no effect on the ventricular pacing threshold in human beings, it was associated with a decrease in post-shock monophasic action potential duration and ventricular effective refractory period, contrary to some previously reported findings.

Determination of ventricular vulnerable period and ventricular fibrillation threshold by use of T-wave shocks in patients undergoing implantation of cardioverter/defibrillators.

BACKGROUND: This study was designed to characterize the ventricular vulnerable period (VVP) and ventricular fibrillation (VF) threshold by use of T-wave shocks in patients undergoing implantation of cardioverter/defibrillators. A premature condensed shock applied during the VVP can induce VF. Most studies on the VVP and VF threshold have been conducted in animals rather than in humans. METHODS AND RESULTS: Twenty-one patients undergoing implantation of Medtronic PCD Jewel 7219D cardioverter/defibrillators because of ventricular tachycardia and/or VF were enrolled. All had structural heart disease. Their ages ranged from 42 to 85 years (mean, 69 +/- 11.3 years). Seventeen (80.9%) had atherosclerotic coronary artery disease. The right ventricle (RV) was driven at a cycle length (S1) of 400 ms, and monophasic shocks (S2) of 0.6 J were delivered through an RV apex lead (cathode) and a superior vena cava lead (anode) during the T wave of each cardiac cycle. The longest and shortest S1-S2 intervals that were capable of inducing sustained VF were defined as the outer and inner limits of the VVP at an energy level of 0.6 J, respectively. To determine the VF threshold, a shock of 0.2 J was applied at the midpoint of the VVP at 0.2-J increments until sustained VF was induced. The lowest energy setting capable of inducing sustained VF was defined as the VF threshold. Of the 21 patients, the VVP at an energy level of 0.6 J averaged 53.8 +/- 26.0 ms. Characteristically, the VVP started from the ascending limb of the T wave and ended at or slightly beyond the peak of the T wave, occupying 12.2 +/- 5.8% of the QT interval. The midpoint of the VVP used for determination of the VF threshold measured 0 to 90 ms (mean, 32.9 +/- 26.0 ms) before the peak of the T wave. Of the 21 patients, 16 (76.2%) had a VF threshold at < or = 0.2 J (estimated 57 V), 3 at 0.4 J (estimated 81 V), and 2 at 0.6 J (estimated 99 V). CONCLUSIONS: The VF threshold is low (< or = 0.2 J) in the majority of patients requiring implantation of cardioverter/defibrillators. Further studies are needed to define clinical usefulness of the study technique relative to its potential role for risk stratification and for assessing antifibrillatory properties of antiarrhythmic drugs in this subset group of patients.

Ventricular fibrillation induced by low-energy shocks from programmable implantable cardioverter-defibrillators in patients with coronary artery disease.

Many of the newest implantable cardioverter-defibrillators (ICDs) provide the option of programmable low-energy cardioversion for monomorphic ventricular tachycardia (VT). Whereas these devices may provide less myocardial damage and increased comfort in patients receiving frequent shocks for VT, the proarrhythmic effects of low-energy cardioversion from ICDs in patients with structural heart disease are not clear. The purpose of this study was to determine prospectively the per-patient incidence of ventricular fibrillation (VF) induction after low-energy cardioversion of VT by ICDs in patients with coronary artery disease. The estimated cardioversion energy requirement was determined during the course of routine predischarge ICD testing in 40 patients with newly implanted ICDs. Two groups of patients were identified during determination of the cardioversion energy requirement: (1) a non-VF group consisting of 26 of 40 patients (65%) without VF induced by low-energy shock and, (2) a VF group consisting of 14 of 40 patients (35%) who developed VF during low-energy cardioversion. There was no difference between the 2 groups in terms of patient age, sex, concurrent antiarrhythmic drug therapy, VT cycle length, or type of ICD system implanted. Compared with the non-VF group, the VF group was more likely to have both a lower ejection fraction (25 +/- 5% vs 33 +/- 8%; p = 0.005) and a cardioversion energy requirement > 2 J (79 vs 27%; p = 0.005). Our results suggest that low-energy cardioversion is associated with a high per-patient risk of VF induction, and the risk is higher in patients with poorer left ventricular function and, possibly, higher cardioversion energy requirement.(ABSTRACT TRUNCATED AT 250 WORDS)

The transcriptional activator Opaque2 recognizes two different target sequences in the 22-kD-like alpha-prolamin genes.

The maize Opaque2 (O2) protein is a "leucine zipper" DNA binding factor that interacts with the sequence TCCACGTAGA in the promoters of the 22-kD alpha-zein genes and activates its transcription. A completely different consensus sequence (GATGAPyPuTGPu) identified in b-32, a gene that encodes an abundant albumin that is also under control of the O2 locus, can also be bound by the O2 protein. We showed that the gene encoding the 22-kD-like alpha-coixin, the alpha-prolamin of the maize-related grass Coix, can also be transactivated by the O2 protein. A binding assay in vitro and footprint analysis demonstrated that the GACATGTC sequence of the alpha-coixin promoter can be recognized and protected by the maize O2 protein. Employing transient expression experiments in immature maize endosperm and tobacco mesophyll protoplasts, we demonstrated that the O2 protein can activate expression of the beta-glucuronidase reporter gene placed under the control of the 22-kD-like alpha-coixin promoter. We also demonstrated that a 22-kD-like alpha-coixin pseudogene promoter is transactivated by the maize O2 protein.

Sequence analysis of 22 kDa-like alpha-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements.

Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis.

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain alpha-, beta-, and gamma-zein and alpha-, beta-, and gamma-coixin. The alpha-coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa alpha-zeins. Like the alpha-zeins, the C1 and C2 alpha-coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to gamma-coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of gamma-zein and represents 15% of the total coixin. The beta-zein fraction was composed of a major 17 kDa protein band, while the beta-coixin fraction consisted of a mixture of alpha- and gamma-coixins. Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa alpha-zein, as did C4 and C5 antisera. The antiserum against gamma-coixin showed strong cross-reaction with gamma-zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa alpha-zeins as well as the 28 and 16 kDa gamma-zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa alpha-zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of zein genes in maize.

The band patterns of zein polypeptides were determined for many commercial inbred corn lines and maize stocks using isofocusing in agarose gels and sodium dodecyl sulfate (SDS)-urea gels. Each inbred line or homozygous maize strain genotype has a distinct zein profile which has been catalogued according to the distance of charge migration and molecular weight (kilodaltons). Several zein polypeptides were mapped to chromosomes 4 and 10 with the use of reciprocal translocations. The mapping of at least two polypeptides on distal 4L and 10L had not been previously reported. The general methods used in the present research will permit the mapping of all the zein polypeptides to chromosomal sites.