An <Emphasis Type="Italic">L</Emphasis><Subscript>1</Subscript>-regularized logistic model for detecting short-term neuronal interactions

Interactions among neurons are a key component of neural signal processing. Rich neural data sets potentially containing evidence of interactions can now be collected readily in the laboratory, but existing analysis methods are often not sufficiently sensitive and specific to reveal these interactions. Generalized linear models offer a platform for analyzing multi-electrode recordings of neuronal spike train data. Here we suggest an L ₁-regularized logistic regression model (L ₁ L method) to detect short-term (order of 3 ms) neuronal interactions. We estimate the parameters in this model using a coordinate descent algorithm, and determine the optimal tuning parameter using a Bayesian Information Criterion. Simulation studies show that in general the L ₁ L method has better sensitivities and specificities than those of the traditional shuffle-corrected cross-correlogram (covariogram) method. The L ₁ L method is able to detect excitatory interactions with both high sensitivity and specificity with reasonably large recordings, even when the magnitude of the interactions is small; similar results hold for inhibition given sufficiently high baseline firing rates. Our study also suggests that the false positives can be further removed by thresholding, because their magnitudes are typically smaller than true interactions. Simulations also show that the L ₁ L method is somewhat robust to partially observed networks. We apply the method to multi-electrode recordings collected in the monkey dorsal premotor cortex (PMd) while the animal prepares to make reaching arm movements. The results show that some neurons interact differently depending on task conditions. The stronger interactions detected with our L ₁ L method were also visible using the covariogram method.     

The multifunctional glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a novel macrophage lactoferrin receptor

Several proteins with limited cell type distribution have been shown to bind lactoferrin. However, except in the case of hepatic and intestinal cells, these have not been definitively identified and characterized. Here we report that the multifunctional glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a novel receptor for lactoferrin (Lf) in macrophages. GAPDH is a well-known moonlighting protein, and previous work from our laboratory has indicated its localization on macrophage cell surfaces, wherein it functions as a transferrin (Tf) receptor. The K(D) value for GAPDH-lactoferrin interaction was determined to be 43.8 nmol/L. Utilizing co-immunoprecipitation, immunoflorescence, and immunogold labelling electron microscopy we could demonstrate the trafficking of lactoferrin to the endosomal compartment along with GAPDH. We also found that upon iron depletion the binding of lactoferrin to macrophage cell surface is enhanced. This correlated with an increased expression of surface GAPDH, while other known lactoferrin receptors CD14 and lipoprotein receptor-related protein (LRP) were found to remain unaltered in expression levels. This suggests that upon iron depletion, cells prefer to use GAPDH to acquire lactoferrin. As GAPDH is an ubiquitously expressed molecule, its function as a receptor for lactoferrin may not be limited to macrophages.     

Epitope enhancement of a CD4 HIV epitope toward the development of the next generation HIV vaccine

Virus-specific CD4+ T cell help and CD8+ cytotoxic T cell responses are critical for maintenance of effective immunity in chronic viral infections. The importance of CD4+ T cells has been documented in HIV infection. To investigate whether a stronger CD4+ T cell response can be induced by modifications to enhance the T1 epitope, the first CD4+ T cell epitope discovered in HIV-1-gp120, we developed a T1-specific CD4+ T cell line from a healthy volunteer immunized with a canarypox vector expressing gp120 and boosted with recombinant gp120. This T1-specific CD4+ T cell line was restricted to DR13, which is common in U.S. Caucasians and African-Americans and very frequent in Africans. Peptides with certain amino acid substitutions in key positions induced enhanced specific CD4+ T cell proliferative responses at lower peptide concentration than the original epitope. This relatively conserved CD4 epitope improved by the epitope enhancement strategy could be a component of a more effective second generation vaccine construct for HIV infection.     

Heat stress induced apoptosis in BC-8 cells derived from AK-5 tumor involves downregulation of Bcl-2 and generation of reactive oxygen species

BC-8, a rat histiocytoma undergoes apoptosis after heat shock, which is due to lack of an effective heat shock response. Heat shock induced generation of free radicals, which in turn are involved in the induction of apoptotic death in BC-8 cells. Treatment of BC-8 cells with N-acetylcysteine partially inhibited the heat induced apoptosis. Introduction of Bcl-2 gene in these cells did not protect them from apoptotic death, whereas transfection with hsp-70 gene did render these cells resistant to heat induced apoptosis transiently. Heat shock also downregulated the expression of Bcl-2 and p53 in these cells. These observations suggested that the heat shock induced apoptosis was mediated through reactive oxygen species and controlled upstream of Bcl-2 check point.     

Models of spatially restricted biochemical reaction systems

Many reactions within the cell occur only in specific intracellular regions. Such local reaction networks give rise to microdomains of activated signaling components. The dynamics of microdomains can be visualized by live cell imaging. Computational models using partial differential equations provide mechanistic insights into the interacting factors that control microdomain dynamics. The mathematical models show that, for membrane-initiated signaling, the ratio of the surface area of the plasma membrane to the volume of the cytoplasm, the topology of the signaling network, the negative regulators, and kinetic properties of key components together define microdomain dynamics. Thus, patterns of locally restricted signaling reaction systems can be considered an emergent property of the cell.     

Mitochondrial DNA Variants of Respiratory Complex I that Uniquely Characterize Haplogroup T2 Are Associated with Increased Risk of Age-Related Macular Degeneration

Background

Age-related macular degeneration (AMD), a chronic neurodegenerative and neovascular retinal disease, is the leading cause of blindness in elderly people of western European origin. While structural and functional alterations in mitochondria (mt) and their metabolites have been implicated in the pathogenesis of chronic neurodegenerative and vascular diseases, the relationship of inherited variants in the mitochondrial genome and mt haplogroup subtypes with advanced AMD has not been reported in large prospective cohorts.

Methodology/Prinicipal Findings

We examined the relationship of inherited mtDNA variants with advanced AMD in 1168 people using a three-stage design on samples from 12-year and 10-year prospective studies on the natural history of age-related eye disease. In Stage I we resequenced the entire genome in 99 elderly AMD-free controls and 215 people with advanced AMD from the 12-year study. A consistent association with AMD in 14 of 17 SNPs characterizing the mtDNA T haplogroup emerged. Further analysis revealed these associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of MT-ND4 and A14233G of MT-ND6). We genotyped T haplogroups in an independent sample of 490 cases and 61 controls from the same study (Stage II) and in 56 cases and 246 controls from the 10-year study (Stage III). People in the T2 haplogroup were approximately 2.5 times more likely to have advanced AMD than their peers (odds ratio [OR] = 2.54, 95%CI 1.36–4.80, P≤0.004) after considering the totality of evidence. Findings persisted after considering the impact of AMD-associated variants A69S and Y402H (OR = 5.19, 95%CI 1.19–22.69, P≤0.029).

Conclusion

Loci defining the mtDNA T2 haplogroup and Complex I are reasonable targets for novel functional analyses and therapeutic research in AMD.     

EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

Age-related cataract is a major cause of blindness worldwide, and cortical cataract is the second most prevalent type of age-related cataract. Although a significant fraction of age-related cataract is heritable, the genetic basis remains to be elucidated. We report that homozygous deletion of Epha2 in two independent strains of mice developed progressive cortical cataract. Retroillumination revealed development of cortical vacuoles at one month of age; visible cataract appeared around three months, which progressed to mature cataract by six months. EPHA2 protein expression in the lens is spatially and temporally regulated. It is low in anterior epithelial cells, upregulated as the cells enter differentiation at the equator, strongly expressed in the cortical fiber cells, but absent in the nuclei. Deletion of Epha2 caused a significant increase in the expression of HSP25 (murine homologue of human HSP27) before the onset of cataract. The overexpressed HSP25 was in an underphosphorylated form, indicating excessive cellular stress and protein misfolding. The orthologous human EPHA2 gene on chromosome 1p36 was tested in three independent worldwide Caucasian populations for allelic association with cortical cataract. Common variants in EPHA2 were found that showed significant association with cortical cataract, and rs6678616 was the most significant in meta-analyses. In addition, we sequenced exons of EPHA2 in linked families and identified a new missense mutation, Arg721Gln, in the protein kinase domain that significantly alters EPHA2 functions in cellular and biochemical assays. Thus, converging evidence from humans and mice suggests that EPHA2 is important in maintaining lens clarity with age.     

Selective release of microRNA species from normal and malignant mammary epithelial cells

MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.     

Deficient innate immunity,thymopoiesis, and gene expression response to radiation in survivors of childhood acute lymphoblastic leukemia

Background: Survivors of childhood acute lymphoblastic leukemia (ALL) are at an increased risk of developing secondary malignant neoplasms. Radiation and chemotherapy can cause mutations and cytogenetic abnormalities and induce genomic instability. Host immunity and appropriate DNA damage responses are critical inhibitors of carcinogenesis. Therefore, we sought to determine the long-term effects of ALL treatment on immune function and response to DNA damage. Methods: Comparative studies on 14 survivors in first complete remission and 16 siblings were conducted. Results: In comparison to siblings on the cells that were involved in adaptive immunity, the patients had either higher numbers (CD19+ B cells and CD4+CD25+ T regulatory cells) or similar numbers (αβT cells and CD45RO+/RA? memory T cells) in the blood. In contrast, patients had lower numbers of all lymphocyte subsets involved in innate immunity (γδT cells and all NK subsets, including KIR2DL1+ cells, KIR2DL2/L3+ cells, and CD16+ cells), and lower natural cytotoxicity against K562 leukemia cells. Thymopoiesis was lower in patients, as demonstrated by less CD45RO?/RA+ naïve T cell and less SjTREC levels in the blood, whereas the Vβ spectratype complexity score was similar. Array of gene expression response to low-dose radiation showed that about 70% of the probesets had a reduced response in patients. One of these genes, SCHIP-1, was also among the top-ranked single nucleotide polymorphisms (SNPs) during the whole-genome scanning by SNP microarray analysis. Conclusion: ALL survivors were deficient in innate immunity, thymopoiesis, and DNA damage responses to radiation. These defects may contribute to their increased likelihood of second malignancy.     

Pleiotropic effects of a chromosome 3 locus on speech-sound disorder and reading

Speech-sound disorder (SSD) is a complex behavioral disorder characterized by speech-sound production errors associated with deficits in articulation, phonological processes, and cognitive linguistic processes. SSD is prevalent in childhood and is comorbid with disorders of language, spelling, and reading disability, or dyslexia. Previous research suggests that developmental problems in domains associated with speech and language acquisition place a child at risk for dyslexia. Recent genetic studies have identified several candidate regions for dyslexia, including one on chromosome 3 segregating in a large Finnish pedigree. To explore common genetic influences on SSD and reading, we examined linkage for several quantitative traits to markers in the pericentrometric region of chromosome 3 in 77 families ascertained through a child with SSD. The quantitative scores measured several processes underlying speech-sound production, including phonological memory, phonological representation, articulation, receptive and expressive vocabulary, and reading decoding and comprehension skills. Model-free linkage analysis was followed by identification of sib pairs with linkage and construction of core shared haplotypes. In our multipoint analyses, measures of phonological memory demonstrated the strongest linkage (marker D3S2465, P=5.6 x 10(-5), and marker D3S3716, P=6.8 x 10(-4)). Tests for single-word decoding also demonstrated linkage (real word reading: marker D3S2465, P=.004; nonsense word reading: marker D3S1595, P=.005). The minimum shared haplotype in sib pairs with similar trait values spans 4.9 cM and is bounded by markers D3S3049 and D3S3045. Our results suggest that domains common to SSD and dyslexia are pleiotropically influenced by a putative quantitative trait locus on chromosome 3.