Quick, practical selection of effective seeds for homology search.

It has been observed that in homology search gapped seeds have better sensitivity than ungapped ones for the same cost (weight). In this paper, we propose a probability leakage model (a dissipative Markov system) to elucidate the mechanism that confers power to spaced seeds. Based on this model, we identify desirable features of gapped search seeds and formulate an extremely efficient procedure for seed design: it samples from the set of spaced seed exhibiting those features, evaluates their sensitivity, and then selects the best. The sensitivity of the constructed seeds is negligibly less than that of the corresponding known optimal seeds. While the challenging mathematical question of characterizing optimal search seeds remains open, we believe that our eminently efficient and effective approach represents a satisfactory solution from a practitioner's viewpoint.     

Recognition of Herpes Simplex Virus Type 2 Tegument Proteins by CD4 T Cells Infiltrating Human Genital Herpes Lesions

The local cellular immune response to herpes simplex virus (HSV) is important in the control of recurrent HSV infection. The antiviral functions of infiltrating CD4-bearing T cells may include cytotoxicity, inhibition of viral growth, lymphokine secretion, and support of humoral and CD8 responses. The antigens recognized by many HSV-specific CD4 T cells localizing to genital HSV-2 lesions are unknown. T cells recognizing antigens encoded within map units 0.67 to 0.73 of HSV DNA are frequently recovered from herpetic lesions. Expression cloning with this region of DNA now shows that tegument protein VP22 and the viral dUTPase, encoded by genes U_L49 and U_L50, respectively, are T-cell antigens. Separate epitopes in VP22 were defined for T-cell clones from each of three patients. Reactivity with the tegument protein encoded by U_L21 was identified for an additional patient. Three new epitopes were identified in VP16, a tegument protein associated with VP22. Some tegument-specific CD4 T-cell clones exhibited cytotoxic activity against HSV-infected cells. These results suggest that herpes simplex tegument proteins are processed for antigen presentation in vivo and are possible candidate compounds for herpes simplex vaccines.     

Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota,USA

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut‐off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi‐locus PCR‐RFLP genotyping using 10 markers (SAG1, SAG2 (5′‐3′SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22‐8, c29‐2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.     

The presence of serotonin in cigarette smoke – a possible mechanistic link to 5-HT-induced airway inflammation

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.     

Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation

Among anti-cancer candidate drugs, TRAIL might be the most specific agent against cancer cells due to its low toxicity to normal cells. Unfortunately, cancer cells usually develop drug resistance to TRAIL, which is a major obstacle for its clinical application. One promising strategy is co-administrating with sensitizer to overcome cancer cells resistance to TRAIL. Clitocine, a natural amino nucleoside purified from wild mushroom, is recently demonstrated that can induce apoptosis in multidrug-resistant human cancer cells by targeting Mcl-1. In the present study,we found that pretreatment with clitocine dramatically enhances TRAIL lethality in its resistant human colon cancer cells by inducing apoptosis. More importantly, combination of clitocine and TRAIL also effectively inhibits xenograft growth and induces tumor cells apoptosis in athymic mice. The disruption of the binding between Mcl-1 and Bak as well as mitochondrial translocation of Bax mediated by clitocine are identified as the key underlying mechanisms, which leading to mitochondrial membrane permeabilization. Enforced exogenous Mcl-1 can effectively attenuate clitocine/TRAIL-induced apoptosis by suppressing the activation of intrinsic apoptotic pathway. Furthermore, clitocine regulates Mcl-1 expression at the posttranslational level as no obvious change is observed on mRNA level and proteasome inhibitor MG132 almost blocks the Mcl-1 suppression by clitocine. In fact, more ubiquitinated Mcl-1 was detected under clitocine treatment. Our findings indicate that clitocine is potentially an effective adjuvant agent in TRAIL-based cancer therapy.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.     

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO₂ concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

A high-throughput system was developed to clone large, complex genes at high frequency and perform mutant complementation and protein tagging with a range of fluorophores in Chlamydomonas reinhardtii.     

Activation of autophagy is involved in the protective effect of 17β‐oestradiol on endotoxaemia‐induced multiple organ dysfunction in ovariectomized rats

Oestrogens have been reported to attenuate acute inflammation in sepsis. In this study, the effects of long‐term oestrogen replacement with 17β‐oestradiol (E₂) on endotoxaemia‐induced circulatory dysfunction and multiple organ dysfunction syndrome were evaluated in ovariectomized (Ovx) rats. E₂ (50 μg/kg, s.c., 3 times/week) was administered for 8 weeks, followed by the induction of endotoxaemia by intravenous infusion of lipopolysaccharides (LPS; 30 mg/kg/4 hrs). Oestrogen deficiency induced by ovariectomy for 9 weeks augmented the LPS‐induced damage, including endotoxic shock, myocardial contractile dysfunction, renal dysfunction and rhabdomyolysis. Cardiac levels of NF‐κB p65, iNOS and oxidized glutathione, free radical production in skeletal muscles, myoglobin deposition in renal tubules, and plasma levels of plasminogen activator inhibitor‐1, TNF‐α, and IL‐6 were more pronounced in the Ovx + LPS group than in the Sham + LPS group. Long‐term treatment of E₂ prevented this amplified damage in Ovx rats. Six hours after LPS initiation, activation of the autophagic process, demonstrated by increases in Atg12 and LC3B‐II/LC3B‐I ratios, and induction of haem oxygenase (HO)‐1 and heat‐shock protein (HSP) 70 protein expression in myocardium were increased significantly in the Ovx + E₂ + LPS group. These results suggest that activation of autophagy and induction of HO‐1 and HSP70 contribute to the protective effect of long‐term E₂ replacement on multiple organ dysfunction syndrome in endotoxaemia.