Enterovirus 70 binds to different glycoconjugates containing alpha2,3-linked sialic acid on different cell lines

Enterovirus 70 (EV70), the causative agent of acute hemorrhagic conjunctivitis, exhibits a restricted tropism for conjunctival and corneal cells in vivo but infects a wide spectrum of mammalian cells in culture. Previously, we demonstrated that human CD55 is a receptor for EV70 on HeLa cells but that EV70 also binds to sialic acid-containing receptors on a variety of other human cell lines. Virus recognition of sialic acid attached to underlying glycans by a particular glycosidic linkage may contribute to host range, tissue tropism, and pathogenesis. Therefore, we tested the possibility that EV70 binds to alpha2,3-linked sialic acid, like other viruses associated with ocular infections. Through the use of linkage-specific sialidases, sialyltransferases, and lectins, we show that EV70 recognizes alpha2,3-linked sialic acid on human corneal epithelial cells and on U-937 cells. Virus attachment to both cell lines is CD55 independent and sensitive to benzyl N-acetyl-alpha-D-galactosaminide, an inhibitor of O-linked glycosylation. Virus binding to corneal cells, but not U-937 cells, is inhibited by proteinase K, but not by phosphatidylinositol-specific phospholipase C treatment. These results are consistent with the idea that a major EV70 receptor on corneal epithelial cells is an O-glycosylated, non-glycosyl phosphatidylinositol-anchored membrane glycoprotein containing alpha2,3-linked sialic acid, while sialylated receptors on U-937 cells are not proteinaceous.     

Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals

Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA. We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DNA substrates were prepared by gamma-irradiation of DNA solutions under N(2)O or air, such that they contained a multiplicity of modified bases. Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases. Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics. The kinetic parameters also depended on the identity of the individual modified base being removed. Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90-->Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study. This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates. The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.     

A modified spray reagent for the detection of amino acids on thin layer chromatography plates

Summary. Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced. Received March 14, 2000 Accepted August 31, 2000     

Bacillus sphaericus interferes with the development of Brugia malayi in Aedes aegypti.

Aedes aegypti (black-eyed Liverpool strain) were exposed to a sublethal dose (LD25) of Bacillus sphaericus and were fed to Mastomys coucha infected with Brugia malayi. The development of the filarial parasite was found to be arrested mostly at the second larval stage. The infection (P < 0.05), infectivity rates (P < 0.001) and L3 load (P < 0.001) were found to be reduced significantly in the treated group.     

Automated Detection of Actinic Keratoses in Clinical Photographs

Background

Clinical diagnosis of actinic keratosis is known to have intra- and inter-observer variability, and there is currently no non-invasive and objective measure to diagnose these lesions.

Objective

The aim of this pilot study was to determine if automatically detecting and circumscribing actinic keratoses in clinical photographs is feasible.

Methods

Photographs of the face and dorsal forearms were acquired in 20 volunteers from two groups: the first with at least on actinic keratosis present on the face and each arm, the second with no actinic keratoses. The photographs were automatically analysed using colour space transforms and morphological features to detect erythema. The automated output was compared with a senior consultant dermatologist’s assessment of the photographs, including the intra-observer variability. Performance was assessed by the correlation between total lesions detected by automated method and dermatologist, and whether the individual lesions detected were in the same location as the dermatologist identified lesions. Additionally, the ability to limit false positives was assessed by automatic assessment of the photographs from the no actinic keratosis group in comparison to the high actinic keratosis group.

Results

The correlation between the automatic and dermatologist counts was 0.62 on the face and 0.51 on the arms, compared to the dermatologist’s intra-observer variation of 0.83 and 0.93 for the same. Sensitivity of automatic detection was 39.5% on the face, 53.1% on the arms. Positive predictive values were 13.9% on the face and 39.8% on the arms. Significantly more lesions (p<0.0001) were detected in the high actinic keratosis group compared to the no actinic keratosis group.

Conclusions

The proposed method was inferior to assessment by the dermatologist in terms of sensitivity and positive predictive value. However, this pilot study used only a single simple feature and was still able to achieve sensitivity of detection of 53.1% on the arms.This suggests that image analysis is a feasible avenue of investigation for overcoming variability in clinical assessment. Future studies should focus on more sophisticated features to improve sensitivity for actinic keratoses without erythema and limit false positives associated with the anatomical structures on the face.     

Effect of amyloids on the vesicular machinery: implications for somatic neurotransmission

Certain neurodegenerative diseases are thought to be initiated by the aggregation of amyloidogenic proteins. However, the mechanism underlying toxicity remains obscure. Most of the suggested mechanisms are generic in nature and do not directly explain the neuron-type specific lesions observed in many of these diseases. Some recent reports suggest that the toxic aggregates impair the synaptic vesicular machinery. This may lead to an understanding of the neuron-type specificity observed in these diseases. A disruption of the vesicular machinery can also be deleterious for extra-synaptic, especially somatic, neurotransmission (common in serotonergic and dopaminergic systems which are specifically affected in Alzheimer''s disease (AD) and Parkinson''s disease (PD), respectively), though this relationship has remained unexplored. In this review, we discuss amyloid-induced damage to the neurotransmitter vesicular machinery, with an eye on the possible implications for somatic exocytosis. We argue that the larger size of the system, and the availability of multi-photon microscopy techniques for directly visualizing monoamines, make the somatic exocytosis machinery a more tractable model for understanding the effect of amyloids on all types of vesicular neurotransmission. Indeed, exploring this neglected connection may not just be important, it may be a more fruitful route for understanding AD and PD.     

AI-supported modified risk staging for multiple myeloma cancer useful in real-world scenario

Introduction: An efficient readily employable risk prognostication method is desirable for MM in settings where genomics tests cannot be performed owing to geographical/economical constraints. In this work, a new Modified Risk Staging (MRS) has been proposed for newly diagnosed Multiple Myeloma (NDMM) that exploits six easy-to-acquire clinical parameters i.e. age, albumin, β2-microglobulin (β2M), calcium, estimated glomerular filtration rate (eGFR) and hemoglobin.Materials and Methods: MRS was designed using a training cohort of 716 NDMM patients of our inhouse MM Indian (MMIn) cohort and validated on MMIn (n=354) cohort and MMRF (n=900) cohort. K-adaptive partitioning (KAP) was used to find new thresholds for the parameters. Risk staging rules, obtained via training a J48 classifier, were used to build MRS.Results: New thresholds were identified for albumin (3.6 g/dL), β2M (4.8 mg/L), calcium (11.13 mg/dL), eGFR (48.1 mL/min), and hemoglobin (12.3 g/dL) using KAP on the MMIn dataset. On the MMIn dataset, MRS outperformed ISS for OS prediction in terms of C-index, hazard ratios, and its corresponding p-values, but performs comparable in prediction of PFS. On both MMIn and MMRF datasets, MRS performed better than RISS in terms of C-index and p-values. A simple online tool was also designed to allow automated calculation of MRS based on the values of the parameters.Discussion: Our proposed ML-derived yet simple staging system, MRS, although does not employ genetic features, outperforms RISS as confirmed by better separability in KM survival curves and higher values of C-index on both MMIn and MMRF datasets.Funding: Grant: BT/MED/30/SP11006/2015 (Department of Biotechnology, Govt. of India), Grant: DST/ICPS/CPS-Individual/2018/279(G) (Department of Science and Technology, Govt. of India), UGC-Senior Research Fellowship.     

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.