A gDNA Microarray for Genotyping Salvia Species

Salvia is an important genus from the Lamiaceae with approximately 1,000 species. This genus is distributed globally and cultivated for ornamental, culinary, and medicinal uses. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia subtracted diversity array (SDA) a suppression subtractive hybridization (SSH) was performed between a pool of Salvia species and a pool of angiosperms and non-angiosperms to selectively isolate Salvia-specific sequences. A total of 285-subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating S. officinalis and S. miltiorrhiza from their closely related species and was also able to reveal genetic relationships consistent with geographical origins. In addition, this approach was capable of isolating highly polymorphic sequences from chloroplast and nuclear DNA without preliminary sequence information. Therefore, SDA is a powerful technique for fingerprinting non-model plants and for identifying new polymorphic loci that may be developed as potential molecular markers.     

Insight from the structural molecular model of cytidylate kinase from Mycobacterium tuberculosis

Mycobacterium tuberculosis is a gram-positive bacterium causes tuberculosis in human. H37Rv strain is a pathogenic strain utilized for tuberculosis research. The cytidylate mono-phosphate (CMP) kinase of Mycobacterium tuberculosis belongs to the family nucleoside mono-phosphate kinase (NMK), this enzyme is required for the bacterial growth. Therefore, it is important to study the structural and functional features of this enzyme in the control of the disease. Hence, we developed the structural molecular model of the CMP kinase protein from Mycobacterium tuberculosis by homology modeling using the software MODELLER (9v10). Based on sequence similarity with protein of known structure (template) of Mycobacterium smegmatis (PDB ID: 3R20) was chosen from protein databank (PDB) by using BLASTp. The energy of constructed models was minimized and the qualities of the models were evaluated by PROCHECK and VERRIFY-3D. Resulted Ramachandran plot analysis showed that conformations for 100.00% of amino acids residues are within the most favored regions. A possible homologous deep cleft active site was identified in the Model using CASTp program. Amino acid composition and polarity of that protein was observed by CLC-Protein Workbench tool. Expasy''s Prot-param server and CYC_REC tool were used for physiochemical and functional characterization of the protein. Studied of secondary structure of that protein was carried out by computational program, ProFunc. The structure is finally submitted in Protein Model Database. The predicted model permits initial inferences about the unexplored 3D structure of the CMP kinase and may be promote in relational designing of molecules for structure-function studies.     

Subtracted Diversity Array Identifies Novel Molecular Markers Including Retrotransposons for Fingerprinting Echinacea Species

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.     

Deferasirox and Vitamin D Improves Overall Survival in Elderly Patients with Acute Myeloid Leukemia after Demethylating Agents Failure

The prognosis of acute myeloid leukemia (AML) in elderly (≥65 years) patients is poor and treatment remains non-consensual especially for those who are not eligible for intensive therapies. Our group has shown that in vitro the iron chelator deferasirox (DFX) synergizes with vitamin D (VD) to promote monocyte differentiation in primary AML cells. Herein, we present results from a retrospective case-control study in which the association of DFX (1–2 g/d) and 25-hydroxycholecalciferol (100,000 IU/week) (DFX/VD) was proposed to patients following demethylating agents failure. Median survival of patients treated with DFX/VD combination (n = 17) was significantly increased in comparison with matched patients receiving best supportive care (BSC) alone (n = 13) (10.4 versus 4 months respectively). In addition, the only factor associated to an increased overall survival in DFX/VD-treated patients was serum VD levels. We conclude that DFX/VD treatment correlated with increased overall survival of AML patients in this retrospective cohort of elderly patients.     

Associative learning in biochemical networks

It has been recently suggested that there are likely generic features characterizing the emergence of systems constructed from the self-organization of self-replicating agents acting under one or more selection pressures. Therefore, structures and behaviors at one length scale may be used to infer analogous structures and behaviors at other length scales. Motivated by this suggestion, we seek to characterize various "animate" behaviors in biochemical networks, and the influence that these behaviors have on genomic evolution. Specifically, in this paper, we develop a simple, chemostat-based model illustrating how a process analogous to associative learning can occur in a biochemical network. Associative learning is a form of learning whereby a system "learns" to associate two stimuli with one another. Associative learning, also known as conditioning, is believed to be a powerful learning process at work in the brain (associative learning is essentially "learning by analogy"). In our model, two types of replicating molecules, denoted as A and B, are present in some initial concentration in the chemostat. Molecules A and B are stimulated to replicate by some growth factors, denoted as G(A) and G(B), respectively. It is also assumed that A and B can covalently link, and that the conjugated molecule can be stimulated by either the G(A) or G(B) growth factors (and can be degraded). We show that, if the chemostat is stimulated by both growth factors for a certain time, followed by a time gap during which the chemostat is not stimulated at all, and if the chemostat is then stimulated again by only one of the growth factors, then there will be a transient increase in the number of molecules activated by the other growth factor. Therefore, the chemostat bears the imprint of earlier, simultaneous stimulation with both growth factors, which is indicative of associative learning. It is interesting to note that the dynamics of our model is consistent with certain aspects of Pavlov's original series of conditioning experiments in dogs. We discuss how associative learning can potentially be performed in vitro within RNA, DNA, or peptide networks. We also describe how such a mechanism could be involved in genomic evolution, and suggest relevant bioinformatics studies that could potentially resolve these issues.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

CD20-specific antibody-targeted chemotherapy of non-Hodgkin’s B-cell lymphoma using calicheamicin-conjugated rituximab

Tumor-targeted delivery of a potent cytotoxic agent, calicheamicin, using its immunoconjugates is a clinically validated therapeutic strategy. Rituximab is a human CD20-specific chimeric antibody extensively used in B-NHL therapy. We investigated whether conjugation to calicheamicin can improve the anti-tumor activity of rituximab against human B-cell lymphoma (BCL) xenografts in preclinical models. BCL cells were cultured with rituximab or its calicheamicin conjugates and their in vitro growth was monitored. BCL cells were injected s.c. to establish localized xenografts in nude mice or i.v. to establish disseminated BCL in severe combined immunodeficient (scid) mice. I.p. treatment with rituximab or its calicheamicin conjugates was initiated and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Conjugation of calicheamicin to rituximab vastly enhanced its growth inhibitory activity against BCL in vitro. Conjugation to calicheamicin had no deleterious effect on the effector functional activity of rituximab. Calicheamicin conjugated to rituximab with an acid-labile linker exhibited greater anti-tumor activity against s.c. BCL xenografts and improved survival of mice with disseminated BCL over that of unconjugated rituximab. Anti-tumor activities of rituximab conjugated to calicheamicin via an acid-stable linker were similar to that of unconjugated rituximab. Superior anti-tumor efficacy exhibited by a calicheamicin immunoconjugate of rituximab with an acid-labile linker over that of rituximab demonstrates the therapeutic potential of CD20-specific antibody-targeted chemotherapy strategy in the treatment of B-NHL. All authors are employed by Wyeth Research.