Circulating Serum MicroRNA-130a as a Novel Putative Marker of Extramedullary Myeloma

Poor outcome of extramedullary disease in multiple myeloma patients and lack of outcome predictors prompt continued search for new markers of the disease. In this report, we show circulating microRNA distinguishing multiple myeloma patients with extramedullary disease from myeloma patients without such manifestation and from healthy donors. MicroRNA-130a was identified by TaqMan Low Density Arrays and verified by quantitative PCR on 144 serum samples (59 multiple myeloma, 55 myeloma with extramedullary disease, 30 healthy donors) in test and validation cohorts as being down-regulated in myeloma patients with extramedullary disease. Circulating microRNA-130a distinguished myeloma patients with extramedullary disease from healthy donors with specificity of 90.0% and sensitivity of 77.1%, patients with extramedullary disease from newly diagnosed multiple myeloma patients with specificity of 77.1% and sensitivity of 34.3% in the test cohort and with specificity of 91.7% and sensitivity of 30.0% in the validation cohort of patients. Circulating microRNA-130a in patients with extramedullary myeloma was associated with bone marrow plasma cells infiltration. Further, microRNA-130a was decreased in bone marrow plasma cells obtained from patients with extramedullary myeloma in comparison to bone marrow plasma cells of myeloma patients without such manifestation, but it was increased in tumor site plasma cells of patients with extramedullary disease compared to bone marrow plasma cells of such patients (p<0.0001). Together, our data suggest connection between lower level of microRNA-130a and extramedullary disease and prompt further work to evaluate this miRNA as a marker of extramedullary disease in multiple myeloma.     

In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.     

Chronic Social Isolation Compromises the Activity of Both Glutathione Peroxidase and Catalase in Hippocampus of Male Wistar Rats

Chronic neuroendocrine stress usually leads to the elevation of the stress hormones and increased metabolic rate, which is frequently accompanied by oxidative damage to the CNS. In the present study we hypothesized that chronic psychosocial isolation (CPSI) of male Wistar rats, characterized by decreased serum corticosterone (CORT), unaltered catecholamines (CTs), and low blood glucose (GLU), may also promote oxidative imbalance in the CNS, by targeting antioxidant defense system. To test it, we have examined the relation between these input signals and protein expression/activity of antioxidant enzymes (AOEs): superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GLR) in the hippocampus (HIPPO) of CPSI animals. We found that CPSI did not affect SODs or CAT, but decreased activity of GPx and compromised GLR, an enzyme highly dependent on blood GLU for its substrate precursor. Further, we have tested whether the CPSI experience altered AOEs response to a novelty stress, and found that it attenuated peroxide-metabolizing enzymes, CAT and GPx, and decreased GLR activity, even though blood GLU was restored. The altered ratios of hippocampal AOEs in CPSI animals, which were worsened under the combined stress conditions, may lead to the accumulation of peroxide products and oxidative imbalance. The mechanism by which CPSI generate oxidative imbalance in the HIPPO is most likely based on poor systemic energy conditions set by this stress. Such conditions may cause functional decline of CNS structures, such as HIPPO, and are likely to promote state linked to onset of many mood disorders.     

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase,a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.The ergot fungus Claviceps purpurea is a phytopathogenic ascomycete which infects the ears of several grasses, replacing the ovary and producing a hibernation structure, the so-called sclerotium, in which the ergot alkaloids are formed. These substances show a high level of structural homology to some neurotransmitters like serotonin and dopamine and can therefore bind to the same receptors in the central nervous system (CNS), which is the basis for the application of ergot alkaloids in a variety of clinical conditions (15).The biochemistry of ergot alkaloid biosynthesis was first investigated by isolation of intermediates and postulation of a hypothetical pathway as well as enzymes needed for the successive biosynthetic steps of the production (Fig. ​(Fig.1).1). Most of the data were collected by pursuing the fate of radiolabeled precursors in feeding experiments (4). The first enzyme which could be assigned to alkaloid production was dimethylallyltryptophan synthetase (DMATS), which is the key enzyme of the pathway and is encoded by the gene dmaW (18). These analyses were performed with a Claviceps fusiformis strain, but a homolog of dmaW ({"type":"entrez-nucleotide","attrs":{"text":"AY259840","term_id":"32402653","term_text":"AY259840"}}AY259840) possessing a similar function could also be isolated in C. purpurea, as was confirmed by a knockout approach (N. Lorenz and P. Tudzynski, unpublished data). Using genome walking combined with cDNA screening, a 68.5-kb genomic region surrounding dmaW could be sequenced and revealed 14 open reading frames (ORFs) (putative genes) encoding, among others, nonribosomal peptide synthetases (NRPSs), a putative catalase, a CYP450-1 monooxygenase, a putative methyltransferase, and several oxidoreductases (6, 13, 19) (Fig. ​(Fig.2).2). Some of these genes were functionally and biochemically analyzed by a gene replacement approach which revealed their function within the pathway (2, 5, 7). However, there is still a deficit in functional analyses, especially with respect to the early steps within this pathway. The conversion from N-methyl-dimethylallyltryptophan (Me-DMAT) to agroclavine via chanoclavine I and chanoclavine I aldehyde includes successive oxidation and reduction steps mediated by a specific class of enzymes, the oxidoreductases (15) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Biosynthetic pathway of the ergot alkaloid biosynthesis of C. purpurea. Genes analyzed so far have been assigned to the corresponding enzyme at the corresponding position within the pathway. DMAPP, dimethylallyldiphosphate; DMAT, dimethylallyltryptophan; Me-DMAT, N-methyl-DMAT. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)Open in a separate windowFIG. 2.Alkaloid biosynthesis gene cluster of C. purpurea. Highlighted in white is the gene of interest ccsA. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)These enzymes are involved in the biosynthesis of many fungal secondary metabolites. A prominent example is the family of the cytochrome P450 monooxygenases (named after the characteristic peak of 450 nm when complexed with carbon monoxide). Cytochrome P450 (CYP450) monooxygenases catalyze the transfer of one oxygen atom from molecular oxygen to various substrates, mostly accomplished by the involvement of NAD(P)H as an electron donor. The eas cluster of C. purpurea also includes a gene encoding a CYP450 monooxygenase: cloA is involved in the oxidation of elymoclavine, leading to the formation of paspalic acid (7).No further monooxygenase-encoding genes seem to be present in the eas cluster, but several genes code for putative oxidoreductases (easA, easD, easE, easG, and easH). These oxidoreductases are most likely involved in the early steps within the pathway, but none of them has been functionally analyzed so far (15).We initiated a functional analysis of the putative oxidoreductase-encoding gene ccsA (formerly easE) (Fig. ​(Fig.2).2). The coding region of ccsA ({"type":"entrez-nucleotide","attrs":{"text":"AJ011965","term_id":"4499842","term_text":"AJ011965"}}AJ011965; 1,503 bp) is composed of two exons interrupted by an intron of 52 bp, yielding a coding capacity of 483 amino acids (aa). The gene product shows highest similarity to putative oxidoreductases of other ergot alkaloid-producing fungi: EasE of C. fusiformis (e⁻¹⁶⁰; {"type":"entrez-protein","attrs":{"text":"ABV57823","term_id":"157488556","term_text":"ABV57823"}}ABV57823), EasE of Neotyphodium lolii (e⁻¹¹⁸; {"type":"entrez-protein","attrs":{"text":"ABM91450","term_id":"124110194","term_text":"ABM91450"}}ABM91450) and CpoX1 of Aspergillus fumigatus (e⁻⁹⁶; {"type":"entrez-nucleotide","attrs":{"text":"XM_751049","term_id":"71002921","term_text":"XM_751049"}}XM_751049). Analyses of the protein sequence using the program PROSITE revealed a flavin adenine dinucleotide (FAD)-binding domain (pfam01565) spanning the region from amino acids 14 to 161 and a berberine bridge enzyme domain (BBE domain; pfam08031) from amino acids 412 to 457. The role of CcsA in the alkaloid biosynthesis pathway was investigated by knockout of the corresponding gene, followed by functional and biochemical analyses of the deletion mutants.     

Anatrichus bimaculatus sp. n. (Diptera: Chloropidae) and differential characters between A. bimaculatus sp. n. and A. erinaceus

A new species, Anatrichus bimaculatus sp. n. (Diptera: Chloropidae), is described from Zambia. Two African Anatrichus species, A. bimaculatus sp. n. and A. erinaceus, are characterized, and the main differential features are illustrated.     

Factors influencing the production of stilbenes by the knotweed, <Emphasis Type="Italic">Reynoutria</Emphasis> × <Emphasis Type="Italic">bohemica</Emphasis>

Background  

Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria × bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria × bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria × bohemica on clayish substrates and on the production of stilbenes and emodin in this plant.     

Capillary isoelectric focusing--useful tool for detection of the biofilm formation in Staphylococcus epidermidis

The biofilm formation is an important factor of S. epidermidis virulence. Biofilm-positive strains might be clinically more important than biofilm-negative ones. Unlike biofilm-negative staphylococci, biofilm-positive staphylococci are surrounded with an extracellular polysaccharide substance. The presence of this substance on the surface can affect physico-chemical properties of the bacterial cell, including surface charge. 73 S. epidermidis strains were examined for the presence of ica operon, for the ability to form biofilm by Christensen test tube method and for the production of slime by Congo red agar method. Isoelectric points (pI) of these strains were determined by means of Capillary Isoelectric Focusing. The biofilm negative strains focused near pI value 2.3, while the pI values of the biofilm positive strains were near 2.6. Isoelectric point is a useful criterion for the differentiation between biofilm-positive and biofilm-negative S. epidermidis strains.     

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.