Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes. The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Here, the authors study an A. fumigatus enzyme that deacetylates GAG in a metal-dependent manner and constitutes a founding member of a new carbohydrate esterase family.Bio-organic Synthesi

Human Telomerase Reverse Transcriptase (hTERT) Q169 Is Essential for Telomerase Function In Vitro and In Vivo

BACKGROUND:Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS:To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE:We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention

Objective and subjective assessment of sleep in chronic low back pain patients compared with healthy age and gender matched controls: a pilot study

<p>Abstract</p> <p>Background</p> <p>While approximately 70% of chronic low back pain (CLBP) sufferers complain of sleep disturbance, current literature is based on self report measures which can be prone to bias and no objective data of sleep quality, based exclusively on CLBP are available. In accordance with the recommendations of The American Sleep Academy, when measuring sleep, both subjective and objective assessments should be considered as the two are only modestly correlated, suggesting that each modality assesses different aspects of an individual's sleep experience. Therefore, the purpose of this study was to expand previous research into sleep disturbance in CLBP by comparing objective and subjective sleep quality in participants with CLBP and healthy age and gender matched controls, to identify correlates of poor sleep and to test logistics and gather information prior to a larger study.</p> <p>Methods</p> <p>15 CLBP participants (mean age = 43.8 years (SD = 11.5), 53% female) and 15 healthy controls (mean age = 41.5 years (SD = 10.6), 53% female) consented. All participants completed the Pittsburgh Sleep Quality Index, Insomnia Severity Index, Pittsburgh Sleep Diary and the SF36v2. CLBP participants also completed the Oswestry Disability Index. Sleep patterns were assessed over three consecutive nights using actigraphy. Total sleep time (TST), sleep efficiency (SE), sleep latency onset (SL) and number of awakenings after sleep onset (WASO) were derived. Statistical analysis was conducted using unrelated t-tests and Pearson's product moment correlation co-efficients.</p> <p>Results</p> <p>CLBP participants demonstrated significantly poorer overall sleep both objectively and subjectively. They demonstrated lower actigraphic SE (p = .002) and increased WASO (p = .027) but no significant differences were found in TST (p = .43) or SL (p = .97). Subjectively, they reported increased insomnia (p =< .001), lower SE (p =< .001) and increased SL (p =< .001) but no difference between TST (p = .827) and WASO (p = .055). Statistically significant associations were found between low back pain (p = .021, r = -.589), physical health (p = .003, r = -.713), disability levels (p = .025, r = .576), and subjective sleep quality in the CLBP participants but not with actigraphy.</p> <p>Conclusion</p> <p>CLBP participants demonstrated poorer overall sleep, increased insomnia symptoms and less efficient sleep. Further investigation using a larger sample size and a longer period of sleep monitoring is ongoing.</p

Complete Mitochondrial Genome Sequence of Three Tetrahymena Species Reveals Mutation Hot Spots and Accelerated Nonsynonymous Substitutions in Ymf Genes

The ciliate Tetrahymena, a model organism, contains divergent mitochondrial (Mt) genome with unusual properties, where half of its 44 genes still remain without a definitive function. These genes could be categorized into two major groups of KPC (known protein coding) and Ymf (genes without an identified function). To gain insights into the mechanisms underlying gene divergence and molecular evolution of Tetrahymena (T.) Mt genomes, we sequenced three Mt genomes of T.paravorax, T.pigmentosa, and T.malaccensis. These genomes were aligned and the analyses were carried out using several programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (ω) on individual codon sites and via a sliding window approach. Comparative genomic analysis indicated a conserved putative transcription control sequence, a GC box, in a region where presumably transcription and replication initiate. We also found distinct features in Mt genome of T.paravorax despite similar genome organization among these ∼47 kb long linear genomes. Another significant finding was the presence of at least one or more highly variable regions in Ymf genes where majority of substitutions were concentrated. These regions were mutation hotspots where elevated distances and the dn/ds ratios were primarily due to an increase in the number of nonsynonymous substitutions, suggesting relaxed selective constraint. However, in a few Ymf genes, accelerated rates of nonsynonymous substitutions may be due to positive selection. Similarly, on protein level the majority of amino acid replacements occurred in these regions. Ymf genes comprise half of the genes in Tetrahymena Mt genomes, so understanding why they have not been assigned definitive functions is an important aspect of molecular evolution. Importantly, nucleotide substitution types and rates suggest possible reasons for not being able to find homologues for Ymf genes. Additionally, comparative genomic analysis of complete Mt genomes is essential in identifying biologically significant motifs such as control regions

Telomerase activity in melanoma and non-melanoma skin cancer

Telomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen's disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found

Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment

BACKGROUND: Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential. METHODS: We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay. RESULTS: We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices. CONCLUSION: We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue

Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma Cells

<p>Abstract</p> <p>Background</p> <p>Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation.</p> <p>Methods</p> <p>This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively.</p> <p>Results</p> <p>Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells.</p> <p>Conclusion</p> <p>These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.</p

Telomere Shortening Impairs Regeneration of the Olfactory Epithelium in Response to Injury but Not Under Homeostatic Conditions

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc−/−) with short telomeres compared to wild type mice (mTerc+/+) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc−/− mice compared to mTerc+/+ mice. Seven days after chemical induced damage, G3 mTerc−/− mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc+/+ mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people