Value of antibodies to free light chains in immunoperoxidase studies of renal biopsies

Aims Because immunoglobulin abnormalities may affect the kidney, investigation of renal biopsies requires immunohistological study of light chains. A problem is that most antibodies to light chains react with whole immunoglobulins as well as free light chains, and there are generally many more whole immunoglobulins than free light chains. The usefulness of antibodies that only detected free light chains was investigated. Methods Antibodies to free light chains were used in an immunoperoxidase method on paraffin sections of 198 renal biopsies, and compared with conventional antibodies against light chains examined by immunofluorescence on 13 frozen sections and by immunoperoxidase on 46 paraffin sections. Results Immunofluorescence and immunoperoxidase were concordant on 10 of 13 biopsies. Immunofluorescence detected slight deposition of light chains in three biopsies not shown by immunoperoxidase, of undetermined clinical significance. Using immunoperoxidase, the free light chain antibodies were more sensitive than conventional antibodies, giving much cleaner staining and better detection of deposits in AL amyloid, light chain deposition disease and cryoglobulinaemic glomerulonephritis. The free light chain antibodies showed discordance or ambiguity between immunohistological and clinical findings in seven (4%) of 185 patients with known immunoglobulin status. These included two of 28 cases of AL amyloid that showed no light chain deposition. The method was not designed for detection of light chain restriction in neoplastic plasma or lymphoplasmacytic cells. Conclusions Polyclonal antibodies to free light chains are an improvement on conventional antibodies in immunoperoxidase study of paraffin sections of renal biopsies and are useful in everyday practice

Recursive construction of perfect DNA molecules from imperfect oligonucleotides

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms

Serum free light chain measurement aids the diagnosis of myeloma in patients with severe renal failure

<p>Abstract</p> <p>Background</p> <p>Monoclonal free light chains (FLCs) frequently cause rapidly progressive renal failure in patients with multiple myeloma. Immunoassays which provide quantitative measurement of FLCs in serum, have now been adopted into screening algorithms for multiple myeloma and other lymphoproliferative disorders. The assays indicate monoclonal FLC production by the presence of an abnormal κ to λ FLC ratio (reference range 0.26–1.65). Previous work, however, has demonstrated that in patients with renal failure the FLC ratio can be increased above normal with no other evidence of monoclonal proteins suggesting that in this population the range should be extended (reference range 0.37–3.1). This study evaluated the diagnostic sensitivity and specificity of the immunoassays in patients with severe renal failure.</p> <p>Methods</p> <p>Sera from 142 patients with new dialysis-dependent renal failure were assessed by serum protein electrophoresis (SPE), FLC immunoassays and immunofixation electrophoresis. The sensitivity and specificity of the FLC ratio's published reference range was compared with the modified renal reference range for identifying patients with multiple myeloma; by receiver operating characteristic curve analysis.</p> <p>Results</p> <p>Forty one patients had a clinical diagnosis of multiple myeloma; all of these patients had abnormal serum FLC ratios. The modified FLC ratio range increased the specificity of the assays (from 93% to 99%), with no loss of sensitivity. Monoclonal FLCs were identified in the urine from 23 of 24 patients assessed.</p> <p>Conclusion</p> <p>Measurement of serum FLC concentrations and calculation of the serum κ/λ ratio is a convenient, sensitive and specific method for identifying monoclonal FLC production in patients with multiple myeloma and acute renal failure. Rapid diagnosis in these patients will allow early initiation of disease specific treatment, such as chemotherapy plus or minus therapies for direct removal of FLCs.</p

Particulate air pollution and chronic ischemic heart disease in the eastern United States: a county level ecological study using satellite aerosol data

<p>Abstract</p> <p>Background</p> <p>There are several known factors that cause ischemic heart disease. However, the part played by air pollution still remains something of a mystery. Recent attention has focused on the chronic effect of particulate matter on heart disease. Satellite-derived aerosol optical depth (AOD) was found to be correlated with <it>PM</it>_2.5in the eastern US. The objective of this study was to examine if there is an association between aerosol air pollution as indicated by AOD and chronic ischemic heart disease (CIHD) in the eastern US.</p> <p>Methods</p> <p>An ecological geographic study method was employed. Race and age standardized mortality rate (SMR) of CIHD was computed for each of the 2306 counties for the time period 2003–2004. A mean AOD raster grid for the same period was derived from Moderate Resolution Imaging Spectrometer (MODIS) aerosol data and the average AOD was calculated for each county. A bivariate Moran's I scatter plot, a map of local indicator of spatial association (LISA) clusters, and three regression models (ordinary least square, spatial lag, and spatial error) were used to analyze the relationship between AOD and CIHD SMR.</p> <p>Results</p> <p>The global Moran's I value is 0.2673 (<it>p </it>= 0.001), indicating an overall positive spatial correlation of CIHD SMR and AOD. The entire study area is dominated by spatial clusters of AOD against SMR (high AOD and high SMR in the east, and low AOD and low SMR in the west) (permutations = 999, <it>p </it>= 0.05). Of the three regression models, the spatial error model achieved the best fit (R²= 0.28). The effect of AOD is positive and significant (beta = 0.7774, p = 0.01).</p> <p>Conclusion</p> <p>Aerosol particle pollution has adverse effect on CIHD mortality risk in the eastern US. High risk of CIHD mortality was found in areas with elevated levels of outdoor aerosol air pollution as indicated by satellite derived AOD. The evidence of the association would support targeting of policy interventions on such areas to reduce air pollution levels. Remote sensing AOD data could be used as an alternative health-related indictor of air quality.</p

Increased ventral striatal volume in college-aged binge drinkers

BACKGROUND Binge drinking is a serious public health issue associated with cognitive, physiological, and anatomical differences from healthy individuals. No studies, however, have reported subcortical grey matter differences in this population. To address this, we compared the grey matter volumes of college-age binge drinkers and healthy controls, focusing on the ventral striatum, hippocampus and amygdala. METHOD T1-weighted images of 19 binge drinkers and 19 healthy volunteers were analyzed using voxel-based morphometry. Structural data were also covaried with Alcohol Use Disorders Identification Test (AUDIT) scores. Cluster-extent threshold and small volume corrections were both used to analyze imaging data. RESULTS Binge drinkers had significantly larger ventral striatal grey matter volumes compared to controls. There were no between group differences in hippocampal or amygdalar volume. Ventral striatal, amygdalar, and hippocampal volumes were also negatively related to AUDIT scores across groups. CONCLUSIONS Our findings stand in contrast to the lower ventral striatal volume previously observed in more severe forms of alcohol use disorders, suggesting that college-age binge drinkers may represent a distinct population from those groups. These findings may instead represent early sequelae, compensatory effects of repeated binge and withdrawal, or an endophenotypic risk factor

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs

Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site

Lentiviral vectors (LVs) are powerful tools for transgene expression in vivo and in vitro. However, the construction of LVs is of low efficiency, due to the large sizes and lack of proper clone sites. Therefore, it is critical to develop efficient strategies for cloning LVs. Here, we reported a combinatorial strategy to efficiently construct LVs using EGFP, hPlk2 wild type (WT) and mutant genes as inserts. Firstly, site-directed mutagenesis (SDM) was performed to create BamH I site for the inserts; secondly, pWPI LV was dephosphorylated after BamH I digestion; finally, the amounts and ratios of the insert and vector DNA were optimized to increase monomeric ligation. Our results showed that the total percentage of positive clones was approximately 48%±7.6%. Using this method, almost all the vectors could be constructed through two or three minipreps. Therefore, our study provided an efficient method for constructing large-size vectors

Genetic inhibition of neurotransmission reveals role of glutamatergic input to dopamine neurons in high-effort behavior

Midbrain dopamine neurons are crucial for many behavioral and cognitive functions. As the major excitatory input, glutamatergic afferents are important for control of the activity and plasticity of dopamine neurons. However, the role of glutamatergic input as a whole onto dopamine neurons remains unclear. Here we developed a mouse line in which glutamatergic inputs onto dopamine neurons are specifically impaired, and utilized this genetic model to directly test the role of glutamatergic inputs in dopamine-related functions. We found that while motor coordination and reward learning were largely unchanged, these animals showed prominent deficits in effort-related behavioral tasks. These results provide genetic evidence that glutamatergic transmission onto dopaminergic neurons underlies incentive motivation, a willingness to exert high levels of effort to obtain reinforcers, and have important implications for understanding the normal function of the midbrain dopamine system.Fil: Hutchison, M. A.. National Institutes of Health; Estados UnidosFil: Gu, X.. National Institutes of Health; Estados UnidosFil: Adrover, Martín Federico. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lee, M. R.. National Institutes of Health; Estados UnidosFil: Hnasko, T. S.. University of California at San Diego; Estados UnidosFil: Alvarez, V. A.. National Institutes of Health; Estados UnidosFil: Lu, W.. National Institutes of Health; Estados Unido

BFAST: An Alignment Tool for Large Scale Genome Resequencing

BACKGROUND:The new generation of massively parallel DNA sequencers, combined with the challenge of whole human genome resequencing, result in the need for rapid and accurate alignment of billions of short DNA sequence reads to a large reference genome. Speed is obviously of great importance, but equally important is maintaining alignment accuracy of short reads, in the 25-100 base range, in the presence of errors and true biological variation. METHODOLOGY:We introduce a new algorithm specifically optimized for this task, as well as a freely available implementation, BFAST, which can align data produced by any of current sequencing platforms, allows for user-customizable levels of speed and accuracy, supports paired end data, and provides for efficient parallel and multi-threaded computation on a computer cluster. The new method is based on creating flexible, efficient whole genome indexes to rapidly map reads to candidate alignment locations, with arbitrary multiple independent indexes allowed to achieve robustness against read errors and sequence variants. The final local alignment uses a Smith-Waterman method, with gaps to support the detection of small indels. CONCLUSIONS:We compare BFAST to a selection of large-scale alignment tools -- BLAT, MAQ, SHRiMP, and SOAP -- in terms of both speed and accuracy, using simulated and real-world datasets. We show BFAST can achieve substantially greater sensitivity of alignment in the context of errors and true variants, especially insertions and deletions, and minimize false mappings, while maintaining adequate speed compared to other current methods. We show BFAST can align the amount of data needed to fully resequence a human genome, one billion reads, with high sensitivity and accuracy, on a modest computer cluster in less than 24 hours. BFAST is available at (http://bfast.sourceforge.net)