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In this paper we present a reliable bidirectional flow DNA amplification microreactor for processing real-world genomic samples. This system shares the low-power thermal responsiveness of a continuous flow reactor with the low surface area to volume ratio character of stationary reactors for reducing surface inhibitory effects. Silanization with dimethyldichlorosilane in combination with dynamic surface passivation was used to enhance PCR compatibility and enable efficient amplification. For real-time fragment amplification monitoring we have implemented an epimodal fluorescent detection capability. The passivated bidirectional flow system was ultrasensitive, achieving an RNase P gene detection limit of 24 human genome copies with a reaction efficiency of 77%. This starts to rival the performance of a conventional real-time PCR instrument with a reaction efficiency of 93% and revitalizes flow-through PCR as a viable component of lab on a chip DNA analysis formats.

Research has been conducted on the ricin A chain, protein toxins entering mammalian cells via endocytosis. The hypothesis that the lysine paucity reduces the chance of ubiquitination and ubiquitin-mediated proteasomal degradation has been investigated and the results are reported.

Research has been conducted on ricin, a heterodimeric protein toxin with a catalytic polypeptide linked to a cell-binding polypeptide by a disulfide bond. The changes in the catalytic polypeptide structure affected by the interaction with the phospholipid vesicles have been investigated and the results indicate that these changes dramatically increase sensitivity to protease digestion.

Ricin A chain (RTA) is a plant-derived ribosome inactivating protein that inhibits protein synthesis by initiating the removal of specific adenine residues from the 28S ribosomal ribonucleic acid (RNA). The substitution of the Arg 180 of RTA with His reduce N-glycosidase activity and cellular toxicity due to the rearrangement of the active site. The activity of His 180 is sterically blocked by a charged hydrogen between Tyr 80 and Glu 177 which leads to the formation of a hydrophobic pocket. The protonation state of the histidine affects the molecular stability of the protein.

The charge difference between the two naturally occurring enzyme variants chloramphenicol acetyltransferase type I and III [CAT(sub I) and CAT(sub III)] is increased, by charge variation of lysyl residues of CAT, by site- directed mutagenesis. The increased charge purifies the CAT(sub I)/CAT(sub III) and CAT(sub III)/CAT(sub III) hybrid trimers. H-195 is involved in the binding of fusidic acid to CAT(sub I). Hybrids with one functional active site per trimer identifies the binding of fusidic acid to CAT(sub I) using specific amino acid residues at each subunit interface.

The magnitude and nature of electrostatic interactions of coenzyme A (CoA) with chloramphenicol acetyltransferase (CAT) were studied by using site-directed mutagenesis, chemical modification, competitive inhibitors and substrate analogues. The results show that the binding of CoA to CAT involves no ion pairs and a few long-range electrostatic interactions, and that CAT recognizes CoA mainly by hydrophobic and polar, but uncharged, interactions.

Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile. [ABSTRACT FROM AUTHOR]

In this paper we present a reliable bidirectional flow DNA amplification microreactor for processing real-world genomic samples. This system shares the low-power thermal responsiveness of a continuous flow reactor with the low surface area to volume ratio character of stationary reactors for reducing surface inhibitory effects. Silanization with dimethyldichiorosilane in combination with dynamic surface passivation was used to enhance PCR compatibility and enable efficient amplification. For real- time fragment amplification monitoring we have linplemented an epimodal fluorescent detection capability. The passivated bidirectional flow system was ultrasensitive, achieving an RNase P gene detection limit of 24 human genome copies with a reaction efficiency of 77%. This starts to rival the performance of a conventional real-time PCR instrument with a reaction efficiency of 93% and revitalizes flow-through PCR as a viable component of lab on a chip DNA analysis formats. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the presence of β-amyloid plaques (Aβ) and neurofibrillary tangles (NFTs) in the brain. The prevalence of the disease is increasing and is expected to reach 141 million cases by 2050. Despite the risk factors associated with the disease, there is no known causative agent for AD. Clinical trials with many drugs have failed over the years, and no therapeutic has been approved for AD. There is increasing evidence that pathogens are found in the brains of AD patients and controls, such as human herpes simplex virus-1 (HSV-1). Given the lack of a human model, the route for pathogen entry into the brain remains open for scrutiny and may include entry via a disturbed blood-brain barrier or the olfactory nasal route. Many factors can contribute to the pathogenicity of HSV-1, such as the ability of HSV-1 to remain latent, tau protein phosphorylation, increased accumulation of Aβ in vivo and in vitro , and repeated cycle of reactivation if immunocompromised. Intriguingly, valacyclovir, a widely used drug for the treatment of HSV-1 and HSV-2 infection, has shown patient improvement in cognition compared to controls in AD clinical studies. We discuss the potential role of HSV-1 in AD pathogenesis and argue for further studies to investigate this relationship.

Lp-PLA 2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA 2 . The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC 50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA 2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.

Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.

Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d ) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.

Small hepatocellular carcinomas (HCCs) can be effectively cured by surgery with good clinical outcomes. However, the conventional AFP marker is ineffective in detecting small tumors. Here we employed a proteomic profiling approach to identify a candidate marker for HCC (

Hepatocellular carcinoma (HCC) is a major liver malignancy possessing a high mortality rate and is particularly prevalent in China and Asia. While surgery is the most effective treatment for liver tumor, about 80% of HCC patients are inoperable at presentation and die early due to late diagnosis. For early cancer detection, we employed a proteomic expression profiling approach to identify biomarkers for early stages of HCC and subsequently assessed the clinical feasibility of a novel marker in plasma. Frozen liver tissues from a retrospective cohort of 75 liver patients (39 HCCs, 20 cirrhosis, and 16 nondiseased subjects) were subjected to proteome-wide expression profiling by 2-DE. MALDI-TOF/TOF was used to identify differentially expressed proteins, which were further confirmed by immunoblotting, qPCR, and immunohistochemistry. Conventional RT-PCR was employed to further analyze the abundance of selected biomarker at mRNA level in a separate cohort of 63 plasma samples (35 HCCs, 16 liver cirrhosis, 12 healthy individuals). We successfully identified lamin B1 (LMNB1) that was significantly upregulated in HCC tumors and present in patients' plasma. LMNB1 functions in nuclear envelope lamina and possesses a transcriptional coregulatory activity having an important role in DNA replication, cellular aging, and stress responses. Clinically, the expression level of lamin B1 correlated positively with tumor stages, tumor sizes, and number of nodules. Our findings further showed elevation of circulating LMNB1 marker in plasma could detect early stages of HCC patients, with 76% sensitivity and 82% specificity. In conclusion, lamin B1 is a clinically useful biomarker for early stages of HCC in tumor tissues and plasma, and warrants further clinical investigation.

Hepatocellular carcinoma (HCC) is an aggressive liver cancer but clinically validated biomarkers that can predict natural history of malignant progression are lacking. The present study explored the proteome-wide patterns of HCC to identify biomarker signature that could distinguish cancerous and nonmalignant liver tissues. A retrospective cohort of 80 HBV-associated HCC was included and both the tumor and adjacent nontumor tissues were subjected to proteome-wide expression profiling by 2-DE method. The subjects were randomly divided into the training (n = 55) and validation (n = 25) subsets, and the data analyzed by classification-and-regression tree algorithm. Protein markers were characterized by MALDI-ToF/MS and confirmed by immunohistochemistry, Western blotting and qPCR assays. Proteomic expression signature composed of six biomarkers (haptoglobin, cytochrome b5, progesterone receptor membrane component 1, heat shock 27 kDa protein 1, lysosomal proteinase cathepsin B, keratin I) was developed as a classifier model for predicting HCC. We further evaluated the model using both leave-one-out procedure and independent validation, and the overall sensitivity and specificity for HCC both are 92.5%, respectively. Clinical correlation analysis revealed that these biomarkers were significantly associated with serum AFP, total protein levels and the Ishak's score. The described model using biomarker signatures could accurately distinguish HCC from nonmalignant tissues, which may also provide hints on how normal hepatocytes are transformed to malignant state during tumor progression.