Compressed indexing and local alignment of DNA

MOTIVATION: Recent experimental studies on compressed indexes (BWT, CSA, FM-index) have confirmed their practicality for indexing very long strings such as the human genome in the main memory. For example, a BWT index for the human genome (with about 3 billion characters) occupies just around 1 G bytes. However, these indexes are designed for exact pattern matching, which is too stringent for biological applications. The demand is often on finding local alignments (pairs of similar substrings with gaps allowed). Without indexing, one can use dynamic programming to find all the local alignments between a text T and a pattern P in O(|T||P|) time, but this would be too slow when the text is of genome scale (e.g. aligning a gene with the human genome would take tens to hundreds of hours). In practice, biologists use heuristic-based software such as BLAST, which is very efficient but does not guarantee to find all local alignments. RESULTS: In this article, we show how to build a software called BWT-SW that exploits a BWT index of a text T to speed up the dynamic programming for finding all local alignments. Experiments reveal that BWT-SW is very efficient (e.g. aligning a pattern of length 3 000 with the human genome takes less than a minute). We have also analyzed BWT-SW mathematically for a simpler similarity model (with gaps disallowed), and we show that the expected running time is O(/T/(0.628)/P/) for random strings. As far as we know, BWT-SW is the first practical tool that can find all local alignments. Yet BWT-SW is not meant to be a replacement of BLAST, as BLAST is still several times faster than BWT-SW for long patterns and BLAST is indeed accurate enough in most cases (we have used BWT-SW to check against the accuracy of BLAST and found that only rarely BLAST would miss some significant alignments). AVAILABILITY: www.cs.hku.hk/~ckwong3/bwtsw CONTACT: twlam@cs.hku.hk.     

Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella

Chloroplast genetic engineering is an environmentally friendly approach, where the foreign integrated gene is often expressed at a higher level than nuclear transformation. The cry1Ab gene was successfully transferred into the cabbage chloroplast genome in this study. The aadA and cry1Ab genes were inserted into the pASCC201 vector and driven by the prrn promoter. The cabbage-specific plastid vectors were transferred into the chloroplasts of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by their resistance to spectinomycin and streptomycin. According to antibiotic selection, the regeneration percentage of the two cabbage cultivars was 4-5%. The results of PCR, Southern, Northern hybridization and western analyses indicated that the aadA and cry1Ab genes were not only successfully integrated into the chloroplast genome, but functionally expressed at the mRNA and protein level. Expression of Cry1Ab protein was detected in the range of 4.8-11.1% of total soluble protein in transgenic mature leaves of the two species. Insecticidal effects on Plutella xylostella were also demonstrated in cry1Ab transformed cabbage. The objectives of this study were to establish a gene transformation system for Brassica chloroplasts, and to study the possibility for insect-resistance in dicot vegetables using chloroplast gene transformation.     

A comparative study on kernel-based probabilistic neural networks for speaker verification

This paper compares kernel-based probabilistic neural networks for speaker verification based on 138 speakers of the YOHO corpus. Experimental evaluations using probabilistic decision-based neural networks (PDBNNs), Gaussian mixture models (GMMs) and elliptical basis function networks (EBFNs) as speaker models were conducted. The original training algorithm of PDBNNs was also modified to make PDBNNs appropriate for speaker verification. Results show that the equal error rate obtained by PDBNNs and GMMs is less than that of EBFNs (0.33% vs. 0.48%), suggesting that GMM- and PDBNN-based speaker models outperform the EBFN ones. This work also finds that the globally supervised learning of PDBNNs is able to find decision thresholds that not only maintain the false acceptance rates to a low level but also reduce their variation, whereas the ad-hoc threshold-determination approach used by the EBFNs and GMMs causes a large variation in the error rates. This property makes the performance of PDBNN-based systems more predictable.     

Detection of endogenous boldenone in the entire male horses

Boldenone (1,2-dehydrotestosterone) is a common veterinary anabolic agent. Its structure is very similar to testosterone. Testosterone is endogenous in the horse, whereas there has been no report concerning the detection of endogenous boldenone. This paper reports the direct observation of sulphate conjugate of boldenone in equine urine from entires. The detection procedures involved solid-phase extraction, immunoaffinity column (IAC) purification, and then LC-MS-MS analysis on a Q-ToF instrument. The identification of boldenone sulphate has provided direct evidence for the endogenous nature of boldenone in entire male horses. Quantification data for the normal level of boldenone in Hong Kong racehorses will also be discussed.     

Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation

In vitro studies have demonstrated that ZNT7 is involved in transporting the cytoplasmic zinc into the Golgi apparatus of the cell for zinc storage or to be incorporated into newly synthesized zinc-requiring enzymes/proteins. To evaluate the physiological role of ZNT7, we created a mouse model of Znt7 deficiency by a gene-trap approach. Znt7-deficient mice were zinc-deficient based on their low zinc content in serum, liver, bone, kidney, and small intestine. In embryonic fibroblasts isolated from Znt7-deficient mice, cellular zinc was approximately 50% that of wild-type controls. Znt7-deficient mice also displayed some classic manifestations of dietary zinc deficiency, such as reduced food intake and poor body weight gain. However, the mutant mice did not show any sign of hair abnormality and dermatitis that are commonly associated with dietary zinc deficiency. A radioactive feeding study suggested that Znt7-deficient mice had reduced zinc absorption in the gut resulting in decreased zinc accumulations in other organs in the body. The poor growth found in Znt7-deficient mice could not be corrected by feeding the mutant mice with a diet containing 6-fold higher zinc (180 mg/kg) than the suggested adequate intake amount (30 mg/kg). Furthermore, the reduced body weight gain of the mutant mice was largely due to the decrease in body fat accumulation. We conclude that ZNT7 has essential functions in dietary zinc absorption and in regulation of body adiposity.     

Neutrophilia and elevated serum cytokines are implicated in glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-alpha manifest a disturbed glucose homeostasis. We hypothesized that disturbed glucose homeostasis might affect myeloid functions. Here, we show that GSD-Ia mice exhibit normal neutrophil activities but have elevated myeloid progenitor cells in the bone marrow and spleen. Interestingly, GSD-Ia mice exhibit a persistent increase in peripheral blood neutrophil counts along with elevated serum levels of granulocyte colony stimulating factor and cytokine-induced neutrophil chemoattractant. Taken together, our results suggest that a loss of glucose homeostasis can compromise the immune system, resulting in neutrophilia. This may explain some of the unexpected clinical manifestations seen in GSD-Ia.     

Exosomes in Inflammation and Inflammatory Disease

Exosomes are a subset of extracellular vesicles released by all cell types and involved in local and systemic intercellular communication. In the past decade, research into exosomes has swelled as their important role in the mediation of health and disease has been increasingly established and acknowledged. Exosomes carry a diverse range of cargo including proteins, nucleic acids, and lipids derived from their parental cell that, when delivered to the recipient cell, can confer pathogenic or therapeutic effects through modulation of immunity and inflammation. In this review, the role of exosomes on mediation of immune and inflammatory responses, and their participation in diseases with a significant inflammatory component is discussed. The considerable potential for exosomes in therapy and diagnosis of inflammatory diseases is also highlighted.     

Relaxation Effect of Abacavir on Rat Basilar Arteries

Background

The use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels.

Methods

The relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5′ nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate.

Results

Abacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5’ nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries.

Conclusion

Abacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may not be related to endothelial dysfunction as abacavir does not impair relaxation of blood vessels. The most likely explanation of increased cardiovascular risk may be increased platelet aggregation as suggested by other studies.     

Effect of NYD-SP27 down-regulation on ATP-induced Ca2+-dependent pancreatic duct anion secretion in cystic fibrosis cells

Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca(2+)-dependent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I(SC)) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl(-) channel blocker, DPC or Glibenclamide (1mM), and inhibitor of Na(+)-K(+)-Cl(-) cotransporter, bumetanide (100 microM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 microM), and Ca(2+)-dependent Cl(-) channel (CaCC) blocker, DIDS (100 microM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 microM) stimulated a fast transient I(SC) increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 microM) reduced the ATP-induced I(SC) response in AT-CF cells by 77.9%. PMA (1 microM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 microM) could inhibit the ATP-induced I(SC) response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca(2+) dependent pancreatic anion secretion.