Non-mydriatic Polaroid photography in screening for diabetic retinopathy: evaluation in a clinical setting

Because of fears that Polaroid colour prints produced with a non-mydriatic fundus camera may not detect important sight threatening lesions in diabetes a study was conducted comparing retinal images obtained on Polaroid prints taken in “field” conditions with those on 35 mm transparencies and fluorescein angiograms. Almost one in five (22/127) Polaroid prints could not be assessed owing to poor quality compared with 3 (2.4%) 35 mm transparencies and 2 (1.6%) fluorescein angiograms. The pick up rate of microaneurysms, haemorrhages, and hard and soft (cotton wool spots) exudates was equivalent for Polaroid prints and 35 mm transparencies of equivalent quality. In two cases with disc new vessels, however, these were not seen on the Polaroid prints.The widespread use of Polaroid colour prints obtained with a non-mydriatic camera without the necessary operative and interpretive skills further limits the usefulness of the technique.     

ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner

Mycobacterium tuberculosis (Mtb) downregulates the surface expression of major histocompatibility class II (MHC II) molecules on macrophages via modulating class II transactivator (CIITA) protein of the host cell. This results in decreased effector function of CD4(+) T cells. In macrophages, CIITA is transcribed by the promoters I (pI) and IV (pIV) and the corresponding gene products are referred to as type I and type IV CIITA, respectively. Earlier studies have mainly focused on CIITA transcribed by pIV; however, these studies also showed that type IV CIITA expression was transient and dispensable for MHC II expression. In the present study, we observed that the Mtb 6-kDa, early secreted antigen (ESAT6) inhibited interferon (IFN)-γ-induced type I as well as type IV CIITA, but, interestingly, inhibition of type I CIITA was found to be independent of Toll-like receptor-2 (TLR2), whereas that of type IV was TLR2 dependent. Moreover, we also present evidence to show that ESAT6-mediated inhibition was regulated via remodeling of the chromatin. We found that ESAT6 caused a decrease in the IFN-γ-stimulated methylation of the histone H3K4, as well as in the levels of histone acetylation at the CIITA pI locus in macrophages. We also found the involvement of mitogen-activated protein kinases ERK1/2 and p38 in the regulation of CIITA by ESAT6. In conclusion, our studies suggest that ESAT6 could inhibit the expression of type I and type IV CIITA through different pathways. Furthermore, ESAT6 could signal through putative receptors other than TLR2, and that the inhibition of IFN-γ-stimulated CIITA by ESAT6 was regulated at the chromatin level.     

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68 ± 0.09) was significantly different from that observed in the strand transfer complex (0.07 ± 0.02). The calculated distances were 46 ± 3 Å and 83 ± 5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ∼ 32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (> 120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.     

The Lysyl Oxidase Pro-peptide Attenuates Fibronectin-mediated Activation of Focal Adhesion Kinase and p130Cas in Breast Cancer Cells

The lysyl oxidase (LOX) gene encodes an enzyme (LOX) critical for extracellular matrix maturation. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. In particular, the pro-peptide domain (LOX-PP) released from the secreted precursor protein (Pro-LOX) was found to inhibit the transformed phenotype of breast, lung, and pancreatic cancer cells. However, the mechanisms of action of LOX-PP remained to be determined. Here, the ability of LOX-PP to attenuate the integrin signaling pathway, which leads to phosphorylation of focal adhesion kinase (FAK), and the activation of its downstream target p130^Cas, was determined. In NF639 breast cancer cells driven by Her-2/neu, which signals via Ras, ectopic Pro-LOX and LOX-PP expression inhibited fibronectin-stimulated protein tyrosine phosphorylation. Importantly, phosphorylation of FAK on Tyr-397 and Tyr-576, and p130^Cas were substantially reduced. The amount of endogenous p130^Cas in the Triton X-100-insoluble protein fraction, and fibronectin-activated haptotaxis were decreased. Interestingly, expression of mature LOX enzyme enhanced fibronectin-stimulated integrin signaling. Of note, treatment with recombinant LOX-PP selectively reduced fibronectin-mediated haptotaxis of NF639, MDA-MB-231, and Hs578T breast cancer cells. Thus, evidence is provided that one mechanism of action of LOX-PP tumor suppression is to block fibronectin-stimulated signaling and cell migration.The lysyl oxidase (LOX)² gene family is comprised of five members LOX, LOXL1, LOXL2, LOXL3, and LOXL4, which encode enzymes that modify extracellular matrix (ECM) proteins to promote their cross-linking and deposition (1). The LOX gene is the best characterized and codes for the synthesis of a secreted 50-kDa glycosylated pro-enzyme (Pro-LOX). Pro-LOX is extracellularly processed by proteolytic cleavage to a mature active 32-kDa enzyme (LOX) and an 18-kDa pro-peptide (LOX-PP) by the procollagen C proteinases bone morphogenic protein-1 (BMP-1), and the related tolloid-like proteins TLL1 and TLL2 (2–4). In murine Pro-LOX, proteolytic processing occurs between amino acids Gly-162 and Asp-163, generating LOX-PP containing 141 amino acids (5). LOX-PP contains two consensus N-glycosylation sites, Asn-91 and Asn-138 (murine sequence) (2) and several O-glycosylation sites.³ LOX-PP does not contain any known protein domains, and structural prediction analysis indicates that LOX-PP assembles as an intrinsically disordered protein (6). Among the LOX family members, the C-terminal ends encode the enzyme domain and are highly conserved, whereas the N-terminal ends that encode the pro-peptide region have variable sequences. Based on structural and sequence similarities of the pro-peptide regions, the LOX family members can be divided into two subgroups: LOXL2, LOXL3, and LOXL4 as one group whose propeptide regions contain four scavenger receptor cysteine-rich domains, and LOX and LOXL1 as a separate group with much simpler and smaller pro-peptide region containing no cysteine residues (reviewed in Ref. 1). In contrast to Pro-LOX, the exact maturation site of Pro-LOXL1 is still unidentified.LOX is essential in the formation of blood vessels and in maintaining their normal characteristics (7–9). Up-regulation of LOX expression has been described in stromal cells that surround ductal breast and broncho-pulmonary carcinomas (10).Expression of the LOX gene was found to inhibit the transforming activity of the Ras oncogene in NIH 3T3 fibroblasts and hence was named the “ras recision” gene (rrg) (11, 12). The LOX gene was shown to inhibit growth in soft agar of NIH 3T3 fibroblasts and to attenuate Ras-mediated activation of phosphatidylinositol 3-kinase (PI3K), Akt, and Erk1/2 kinases and NF-κB activation (13). More recently, the rrg activity was mapped to the 18-kDa LOX-PP. Specifically, LOX-PP was shown to inhibit Ras-mediated transformation of fibroblasts as determined by reduced growth in soft agar, localization of PDK1 to the membrane, and activation of NF-κB (14). Furthermore, the inhibitory effects of LOX-PP on Ras signaling were extended to breast, pancreatic, and lung cancer cells (6, 14, 15). LOX-PP expression in these carcinoma cells reverted Her-2/neu- and Ras-mediated epithelial to mesenchymal transition (EMT), leading to increased expression of E-cadherin and γ-catenin, and reduced levels of Snail, vimentin, and/or BCL-2 (7, 15). Furthermore, LOX-PP expression reduced tumor formation in a xenograft model by Her-2/neu-overexpressing NF639 cells (6).Acquisition of the ability to invade the ECM is essential to EMT. The ECM has multiple mechanical and signaling functions. The ECM defines interfaces between tissues, provides a scaffold for cell traction, and a substrate for cell migration and adhesion. It is composed of a complex of proteins such as collagens, fibronectin, and laminin, which can interact and bind various growth factors (16). Fibronectin is of particular interest because it was recently shown to interact with the C terminus of Pro-LOX (17). Binding of fibronectin to its receptors (e.g. integrins α₅β₁ or α_vβ₁) stimulates the tyrosine phosphorylation of cellular proteins, in particular that of focal adhesion kinase (FAK) (18). Little is known about the mechanism of action of LOX-PP. Here, we have asked whether the tumor suppressor activity of LOX-PP attenuates the activation of the integrin signaling pathway in breast cancer cells. We report that LOX-PP attenuates FAK signaling and activation of its downstream target p130^Cas and is a robust inhibitor of fibronectin-stimulated cell migration.     

Quantitative analysis and engineering of fatty acid biosynthesis in E. coli

Fatty acids are central hydrocarbon intermediates in the biosynthesis of diesel from renewable sources. We have engineered an Escherichia coli cell line that produces 4.5 g/L/day total fatty acid in a fed-batch fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved unpredictable. To develop a more reliable engineering strategy, a cell-free system was developed that enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using this system, the strong dependence of fatty acid synthesis on malonyl-CoA availability and several important phenomena in fatty acid synthesis were verified. Results from this cell-free system were confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and target specific steps for protein and metabolic engineering to enhance the catalytic conversion of glucose into biodiesel.     

CBL Is Frequently Altered in Lung Cancers: Its Relationship to Mutations in MET and EGFR Tyrosine Kinases

Background

Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC.

Methods and Findings

Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility.

Conclusions

Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.     

Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage.

Partial chymotryptic digestion of purified avian myeloblastosis virus alpha beta DNA polymerase resulted in the activation of a Mg2+-dependent DNA endonuclease activity. Incubation of the polymerase-protease mixture in the presence of super-coiled DNA and Mg2+ permitted detection of the cleaved polymerase fragment possessing DNA nicking activity. Protease digestion conditions were established permitting selective cleavage of beta to alpha, which contained DNA polymerase and RNase H activity and to a family of polypeptides ranging in size from 30,000 to 34,000 daltons. These latter beta-unique fragments were purified by polyuridylate-Sepharose 4B chromatography and were shown to contain both DNA binding and DNA endonuclease activities. We have demonstrated that this group of polymerase fragments derived by chymotryptic digestion of alpha beta DNA polymerase is similar to the in vivo-isolated avian myeloblastosis virus p32pol in size, sequence, and DNA endonuclease activity.     

Histopathology of Mycotoxicosis produced in Swiss albino mice by metabolites of some fungal isolates.

Of 199 fungal cultures isolated from some of the common cereals collected from different parts of Uttar Pradesh and Madhya Pradesh, 70 produced toxic metabolites. Of the 70 fungi isolated, 59 produced toxins which caused visible lesions in livers, kidneys, and spleens but did not cause mortality. Toxicity, graded in terms of the mortality rate and the extent of lesions in livers, kidneys, and spleens, was found to be highest in various species of Aspergillus (40%), followed by Chaetomium spp. (31%).     

Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, 29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and 29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.     

Nucleic acid amplification strategies for DNA microarray-based pathogen detection

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.