Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

Functional Recovery Occurs Even After Partial Remyelination of Axon-Meshed Median and Ulnar Nerves in Mice

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory–functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.

     

Exogenous expression of caveolin-1 is sufficient for hepatic stellate cell activation

Caveolin-1 (Cav-1) expression is increased in hepatic stellate cells (HSC) upon liver cirrhosis and it functions as an integral membrane protein of lipid rafts and caveolae that regulates and integrates multiple signals as a platform. This study aimed to evaluate the role of Cav-1 in HSC. Thus, the effects of exogenous expression of Cav-1 in GRX cells, a model of activated HSC, were determined. Here, we demonstrated through evaluating well-known HSC activation markers – such as α-smooth muscle actin, collagen I, and glial fibrillary acidic protein – that up regulation of Cav-1 induced GRX to a more activated phenotype. GRX^EGFP-Cav1 presented an increased migration, an altered adhesion pattern, a reorganization f-actin cytoskeleton, an arrested cell cycle, a modified cellular ultrastructure, and a raised endocytic flux. Based on this, GRX ^EGFP-Cav1 represents a new cellular model that can be an important tool for understanding of events related to HSC activation. Furthermore, our results reinforce the role of Cav-1 as a molecular marker of HSC activation.     

Strombidinopsis jeokjo n. sp. (ciliophora: choreotrichida) from the coastal waters off western Korea: morphology and small subunit ribosomal DNA gene sequence

The planktonic ciliate Strombidinopsis jeokjo n. sp. is described from Quantitative Protargol-Stained (QPS) preparations, and the sequence of the small subunit rDNA (SSU rDNA) from cultured cells is reported. This species is ovoid and bluntly tapered towards the posterior. The ranges (and mean +/- standard deviation, n = 31) of cell length, cell width, and oral diameter of the QPS-stained specimens were 100-190 microm (149 +/- 25), 60-105 microm (79 +/- 13), and 55-80 microm (64 +/- 5), respectively. Fifteen to seventeen external oral polykinetids had oral membranelle cilia 20-35 microm long. Twenty-six to twenty-eight somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 23-44 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-7 microm long. Strombidinopsis jeokjo had two ovoid macronuclei of 25-38 microm x 12-15 microm. When properly aligned, the sequence of the SSU rDNA of S. jeokjo (GenBank Accession No. AJ628250) was approximately 2% different from that of an unidentified Strombidinopsis species (GenBank Accession No. AF399132-AF399135), the closest species in the SSU rDNA sequence.     

Identification and characterization of small RNAs from vernalized<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two major classes of small non-coding RNAs with important roles in the regulation of gene expression, such as mRNA degradation and translational repression, heterochromatin formation, genome defense against transposons and viruses in eukaryotes. MiRNA- and siRNA-directed processes have emerged as a regulatory mechanism for growth and development in both animals and plants. To identify small RNAs that might be involved in vernalization, a process accelerating flowering brought on by a long period of cold, we generated a library of small RNAs from Arabidopsis that had been subject to vernalization. From the analysis of the library, 277 small RNAs were identified. They were distributed throughout all the five chromosomes. While the vast majority of small RNA genes locate on intergenic regions, others locate on repeat-rich regions, centromeric regions, transposon-related genes, and protein-coding genes. Five of them were mapped to convergent overlapping gene pairs. Two-hundred and forty of them were novel endogenous small RNAs that have not been cloned yet from plants grown under normal conditions and other environmental stresses. Seven putative miRNAs were up- or down-regulated by vernalization. In conclusion, many small RNAs were identified from vernalized Arabidopsis and some of these identified small RNAs may play roles in plant responses to vernalization.     

Occurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.     

MUN (MERISTEM UNSTRUCTURED), encoding a SPC24 homolog of NDC80 kinetochore complex,affects development through cell division in Arabidopsis thaliana

Kinetochore, a protein super‐complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured‐1 (mun‐1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun‐1 is a weak allele because of the insertion of T‐DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9‐mediated mutagenesis showed zygotic embryonic lethality similar to nuf2‐1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two‐hybrid assay and in planta co‐immunoprecipitation. MUN is co‐localized at the centromere with HTR12/CENH3, which is a centromere‐specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.