Culturing conditions highly affect DNA methylation and gene expression levels in MCF7 breast cancer cell line

The levels of DNA methylation and their role in gene expression are key factors that could affect diagnosis, prognosis, and treatment options of different diseases. In this study, the methylation levels of 22 genes that are mostly correlated to breast cancer were determined using EpiTect methyl II PCR array. This analysis was performed to determine the effect of cells’ passage number and the use of antibiotics in the culturing media on gene methylation levels in MCF7 cell line. DNA methylation levels of PTGS2, ADAM23, HIC1, and PYCARD were found to be significantly different among different passages. While the DNA methylation levels of CCNA1, RASSF1, and THBS1 were found to be affected by the use of 1% of penicillin/streptomycin in the culture media. Gene expression analysis after demethylation using 5-Aza-2′-deoxycytidine showed that the gene expression levels of the hypermethylated genes varied between different passage numbers. This study shows that the presence of antibiotic within cultured media and cell line’s passage number could greatly affect the methylation levels that need to be considered in future studies on cell lines.     

Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia

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Resveratrol protects mice against SEB‐induced acute lung injury and mortality by miR‐193a modulation that targets TGF‐β signalling

Staphylococcal enterotoxin B (SEB) is a potent superantigen produced by Staphylococcus aureus that triggers a strong immune response, characterized by cytokine storm, multi‐organ failure, and often death. When inhaled, SEB can cause acute lung injury (ALI) and respiratory failure. In this study, we investigated the effect of resveratrol (RES), a phytoallexin, on SEB‐driven ALI and mortality in mice. We used a dual‐exposure model of SEB in C3H/HeJ mice, which caused 100% mortality within the first 5 days of exposure, and treatment with RES resulted in 100% survival of these mice up to 10 days post‐SEB exposure. RES reduced the inflammatory cytokines in the serum and lungs, as well as T cell infiltration into the lungs caused by SEB. Treatment with RES also caused increased production of transforming growth factor‐beta (TGF‐β) in the blood and lungs. RES altered the miRNA profile in the immune cells isolated from the lungs. Of these, miR‐193a was strongly induced by SEB and was down‐regulated by RES treatment. Furthermore, transfection studies and pathway analyses revealed that miR‐193a targeted several molecules involved in TGF‐β signalling (TGFβ2, TGFβR3) and activation of apoptotic pathways death receptor‐6 (DR6). Together, our studies suggest that RES can effectively neutralize SEB‐mediated lung injury and mortality through potential regulation of miRNA that promote anti‐inflammatory activities.     

Hyperelastic modeling of the combined effects of tissue swelling and deformation-related collagen renewal in fibrous soft tissue

A continuum mechanics constitutive model is presented for the interaction between swelling and collagen remodeling in biological soft tissue. The model is inherently two-way: swelling stretches the collagen fibers which affects their rate of degradation—the remodeled fibrous microarchitecture provides selective directional stiffening that causes the swollen tissue to expand more in the unreinforced directions. The constitutive model specifically treats stretch-stabilization wherein the rate of enzymatic-induced degradation of collagen is a decreasing function of fiber stretch. New collagen replacement takes place in a generally swollen environment, and this synthesis is tracked as a function of time by means of a time integration scheme that accounts for the historical sequence of collagen recreation. The model allows for the specification of the collagen pre-stretch at the time of first synthesis, thus allowing for the consideration of either initially limp replacement fiber or initially pre-tensioned replacement fiber. Loading and swelling that occurs on time scales that are commensurate with the natural time scales for fiber degradation and replacement lead to the consideration of time-integral constitutive equations. Loading and swelling that take place on time scales that are very different from that of the remodeling time scales provide a simplified treatment in which there are definite notions of a short-time instantaneous response and also a large-time approach to a steady-state condition of homeostasis.     

Host-pathogen diversity in a wild system: Chondrilla juncea–Puccinia chondrillina

The resistance structure of a Turkish population of the clonal, apomictic composite Chondrilla juncea and the pathotypic structure of a co-occurring population of its obligate rust pathogen, Puccinia chondrillina, was determined by sequential inoculation of 19 host lines with 15 pathogen isolates each derived from single pustules collected from separate plants among the host population. The resultant matrix of resistant and susceptible reactions provides strong circumstantial evidence for a gene-for-gene interaction. Seven distinct pathotypes were detected in the pathogen population. One of these comprised 53% of the population, a second comprised 13%, while the remaining five pathotypes were each detected only once. The host population was similarly diverse, being composed of eight resistance phenotypes, only two of which were represented by more than one host line. Although C. juncea is apomictic, there was only 58% congruence between host resistance and multi-locus isozyme phenotype categories within this population. Pathotypic phenotypes of 13 other isolates of P. chondrillina collected from ten other Turkish and three more distant populations of C. juncea were markedly different from those found in the population studied in detail. There was no obvious relationship between the degree of geographic separation of pathotypes and their ability to attack particular C. juncea lines in this or three other populations represented by single host lines. Received: 10 March 1997 / Accepted: 4 August 1997     

High Mitochondrial DNA Diversity with Little Structure Within and Among Leaf Monkey Populations (Trachypithecus cristatus and Trachypithecus auratus)

We used analyses of mitochondrial DNA restriction site polymorphisms to estimate population genetic structure and phylogenetic relationships among 42 individuals from two Asian leaf monkey species (Trachypithecus auratus and T. cristatus) and to compare them to the geographically proximate species, Presbytis comata. We amplified a 2300-base pair fragment spanning the mitochondrial NADH 3 and NADH 4 genes, including their tRNA flanking subunits, glycine and leucine, and digested it with a battery of 22 restriction endonucleases, yielding 21 unique multienzyme haplotypes and 60 variable restriction sites. Presbytis comata is clearly divergent from both Trachypithecus species. Within the Javan T. auratus, our analysis does not support the distinction of two subspecies currently recognized on the basis of morphological features (Weitzel and Groves, 1985). T. auratus and T. cristatus are not internally monophyletic with respect to each other in our phylogenetic analysis. These results indicate either a recent speciation event with the retention of ancestral polymorphisms or that the two taxa are not separate species. Therefore with respect to conserving genetic diversity within the leaf monkey, we would have to consider T. auratus and T. cristatus as essentially one large polymorphic, conservation unit. However, within that conservation unit, T. auratus of Java represent a separate management unit from T. auratus/T. cristatus of Sumatra and Peninsular Malaysia.     

Capsugenin,a dammarane triterpene from Corchorus capsularis

From the leaves of Corchorus capsularis a new dammarane triterpene glycoside, capsin, has been isolated. Capsin was identified as the 3-glucoside of 20,24-epoxy-3β,12β,25,30-tetrahydroxydammarane from spectral data. Capsin was tentatively assigned the (20S, 24S)-configuration by comparison with data available for similar compounds. One of the oxidation products of the aglycone appears to be a friedo-type derivative, formed by concerted methyl migration on decarboxylation of a C-30 carboxylic acid intermediate.     

Photodynamic therapy for Staphylococcus aureus infected burn wounds in mice.

The rise of multiply antibiotic resistant bacteria has led to searches for novel antimicrobial therapies to treat infections. Photodynamic therapy (PDT) is a potential candidate; it uses the combination of a photosensitizer with visible light to produce reactive oxygen species that lead to cell death. We used PDT mediated by meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin (PTMPP) to treat burn wounds in mice with established Staphylococcus aureus infections The third degree burn wounds were infected with bioluminescent S. aureus. PDT was applied after one day of bacterial growth by adding a 25% DMSO/500 microM PTMPP solution to the wound followed by illumination with red light and periodic imaging of the mice using a sensitive camera to detect the bioluminescence. More than 98% of the bacteria were eradicated after a light dose of 210 J cm(-2) in the presence of PTMPP. However, bacterial re-growth was observed. Light alone or PDT both delayed the wound healing. These data suggest that PDT has the potential to rapidly reduce the bacterial load in infected burns. The treatment needs to be optimized to reduce wound damage and prevent recurrence.     

Hydroxysesamone and 2,3-epoxysesamone from roots of Sesamum indicum.

A red naphthoquinone, named hydroxysesamone, was isolated from the roots of Sesamum indicum together with a known yellow naphthoxirene derivative, 2,3-epoxy-2,3-dihydro-5,8-dihydroxy-2-(3-methyl-2-butenyl)-1,4-naphthoquinone, named 2,3-epoxysesamone. The structure of the naphthoquinone was characterized as 2,5,8-trihydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone on the basis of spectral evidence. Full assignments of NMR resonances of 2,3-epoxysesamone were also confirmed by two-dimensional NMR spectroscopic experiments. Chlorosesamone, hydroxysesamone and 2,3-epoxysesamone all showed antifungal activities toward Cladosporium fulvum.     

In vivo excision and amplification of large segments of the Escherichia coli genome.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.