Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG , due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.MASI is frequently present in mutant  KRAS mutations in cancer cell lines frequently demonstrated complete or relative MASI EGFR gene  GFR gene . Even thFLT3 and WT1 mutations in acute myeloid leukemia JAK2 mutations in myeloproliferative disorders Recent genome-wide approaches, especially high resolution single nucleotide polymorphism (SNP) arrays, enable evaluation of dynamic chromosomal as well as focal changes of CNG and loss of heterogeneity (LOH) with very high resolution. Within a few years, these assays have identified several novel lesions with amplification and/or LOH across several organs EGFR pathway genes, including, EGFR, KRAS, BRAF, and PIK3CA genes, are well-investigated oncogenes in many tumors including lung, colorectal (CRC), and pancreatic cancers (PAC) RAS mutations, including KRAS, are the most frequent oncogenic mutations present in human tumors, detected in about 20% of non-small-cell lung cancer (NSCLC), 40% of CRC and over 90% of PAC BRAF and PIK3CA genes are also activated by mutations in CRC EGFR gene are present in 15–30% of NSCLC while they have been rarely detected in other type of human cancers EGFR CNGs were also reported in NSCLC and may play a role in response and survival to tyrosine kinase inhibitor therapy KRAS CNGs have not been investigated in depth in clinical tumors including NSCLCs. Taken together, the inter-relationship between mutations, CNGs and MASI is complex. The goal of the present study is to better understand the complex inter-relationships between mutations, CNGs and MASI, and to clarify the biological and clinical significance of these oncogenic alterations.TP53, CDKN2A, PTEN, RB1, APC, and SMAD) and five oncogenes  tested in 833 cell lines from the database of the Cancer Genome Project, Sanger Institute, Cambridge, UK (www.sanger.ac.uk). We limited our examination to genes having relatively large numbers of mutations (>30) but also included the EGFR gene (7 mutations) which forms the basis of much of our work. Because of stromal cell contamination in clinical tumor samples, we limited our examination to tumor cell lines. We downloaded the free database of mutational status and zygosity status for each gene (on April 8th 2009). Zygosity status of each mutation was determined at the Institute by manual examination of the sequencing electropherograms . We calculated the frequency of homozygosity for each of the 11 genes and for the entire oncogene or tumor suppressor groups.We queried the zygosity status of 11 well-known and frequently mutated genes including six tumor suppressor genes (We studied 114 tumor cell lines of lung cancer (n = 85), CRC (n = 19) or PAC (n = 10) origin. The details of each line are shown in All cell lines were proven to have individual genetic origins by the Powerplex 1.2 system  and, when available, corresponded with their original profiles as obtained from the ATCC.EGFR or KRAS mutations and survival data were selected from a larger set of previously studied resected NSCLC We studied 393 tumors of NSCLC (n = 333) or CRC (n = 60) origin . DNAs frGenomic DNAs were isolated from cell lines, frozen tumors or paraffin embedded tumors (in 19 cases from Tartu University) by standard phenol-chloroform extraction KRAS (mKRAS), BRAF (mBRAF), PIK3CA (mPIK3CA) and EGFR (mEGFR) genes by direct sequencing as described previously We determined the mutational status of http://www.geospiza.com/finchtv.html) which can provide sharp wave lines without boldness after maximization, 2) pixel based wave peak heights measurement using a desktop ruler software, MB-Ruler (http://www.markus-bader.de/MB-Ruler/), and 3) calculation of mA%. For point mutations, we used the following formula: mA% = Hmut./(Hmut+Hwt) (%), where Hmut is the minimum distance between midpoint of mutant wave line at peak and midpoint of baseline, and Hwt is the minimum distance between midpoint of wild type wave line at peak and midpoint of baseline. For deletion or insertion types of mutations, we used the average of mA% of the first five different waves from the beginning of mutations (We quantified the relative ratios between mutant (mA) and wild type (wA) alleles by direct sequencing to determine the percent of the mutant allele (mA%) by three steps : 1) magnutations . We repeIn order to validate mA% detected by direct sequencing, we constructed each mutant or wild type pCR2.1-TOPO plasmid from cell lines harboring 14 kinds of mutations using TOPO TA cloning kit  and QIAprep Miniprep Kit (QIAGEN). We mixed mutant plasmid with corresponding wild type plasmid at various ratios and amplified the mixed plasmid as a template of PCR using paired primer sets for mutational analyses. PCR products were directly sequenced and the mA% were determined by measurement of sequeincing electropherograms. Finally, we confirmed the linearity between the actual mixed proportion of mutant and wild type plasmids and mA% detected by direct sequencing .PCR products were cloned into pCR2.1-TOPO vector using TOPO TA cloning kit (Invitrogen). About 20 clones (range15–25) were randomly selected for sequencing using either M13 forward primer or corresponding primers of each gene. mA% was calculated as the percentage of mutant clones in the total number cloned.EGFR exon 19 deletion type mutations could be distinguished from wA based on 9 to 12 base pairs differences, overnight digestion of PCR products was needed for point mutations using appropriate enzymes which can specifically digest wild type sequences  assay using Power SYBR® Green PCR Master Mix (Applied Biosystems) as previously reported , made from a mixture of human blood cells from six to eight different donors, as a diploid control. Based on our previous study CNGs of Samples were analyzed using the Genome-Wide Human SNP Array 6.0 platform  according to the manufacturer's directions. GeneChip Command Console Software (GCOS) was used to generate feature extracted intensity (.CEL) files which were subsequently processed using the Birdseed v2 algorithm in Genotyping Console 3.0.2 to create genotype (.chp) call files.Copy number and allele status were determined using Partek Genomics Suite . All CEL files were imported using the same default parameters. Copy number values were generated by normalizing each sample's probe set intensity to that of a reference. For tumors, paired references were used consisting of the normal lung tissue profile matching each patient. For lung cancer cell lines, an unpaired, pooled reference generated from the intensities of all 45 normal lung tissue profiles (those matching the tumors described above) was used. Regions of copy number gain and loss were then statistically detected using the Hidden Markov Model (HMM) based segmentation method of the software package with default parameters and the requirement of at least 50 contiguous probe sets.2 software (http://www.flintbox.com/technology.asp?page=3716) Regions of allelic imbalance were determined using the allele specific copy number (AsCN) function of Partek. For paired analysis, only heterozygous SNPs in the reference  were considered informative and the reference intensity for copy number creation was the allele intensity in the normal sample. In unpaired analysis, this reference intensity was taken as the average allele intensity of all reference  samples that were heterozygous for a given SNP. The ethnicity of all patients is listed in mRNA expression of each mutant gene was evaluated by real-time qPCR of cDNA product. Primer sequencing and PCR conditions are provided in Ras activity was evaluated using Ras GTPase Chemi ELISA  following the manufacturer's protocol. Briefly, cell lysates from cell lines were quantified using BSA Protein Assay Kit . Glutathione-S-transferase (GST) fused to ras-binding-domain (RBD) of Raf which can specifically bind only to activated Ras was coated onto glutathione-coated microplates by a one hour incubation. After washing, equal amounts of cell lysates (45 µg) were applied and incubated for one hour. A primary antibody which can detect H- and K-ras was added and incubated for one hour. An hour incubation with a second antibody conjugated to horseradish peroxidase (HRP) and developing chemiluminescent reagents were used to detect activated Ras binding to the plate. The luminescent intensity, which was inversely proportional to the amount of activated Ras, was read using FLUOstar OPTIMA . Each cell line was tested in duplicate. All values presented are relative light units compared with mean value of two HBEC lines that was arbitrarily assigned a value of one.2 ratios and LOH status for each informative SNP in the tumor samples using dChip software with default settings. Regions of hemizygous deletion (i.e. one copy loss in diploid cells) in each sample were determined by identifying SNPs that displayed copy number loss  with concordant LOH. In order to identify the lost allele in these regions, we then calculated allele-specific intensity ratios using the aroma.affymetrix package in R Tumor samples contain varying numbers of stromal and other non malignant cells that may affect estimates of tumor cell gene copy number and allelic imbalance. To estimate tumor DNA content for clinical samples, we used a method adapted from Weir et al The differences of significance among categorized groups were compared using Chi-square or Fisher's exact tests as appropriate for univariate analyses. Univariate analyses of overall survival (OS) were performed using the Kaplan-Meier method with a log-rank test. All data were analyzed with GraphPad Prism 5 software . All statistical tests were two-sided and probability values <0.05 were defined as being statistically significant.APC, while the five oncogenes also had a relatively high frequency of homozygous mutations (20%). However, the frequency of homozygous mutations varied - being frequent in KRAS or EGFR mutant lines but not with PIK3CA mutations. As shown below, the true incidence of MASI is higher, as the Sanger data base does not have quantitative copy number data for cell lines.For the 11 genes queried in the Sanger database, we identified a total of 1436 mutations . As expePIK3CA to 38% for KRAS. The frequencies of homozygous mutations for EGFR (20%) and BRAF (13%) were intermediate. Thus our findings are similar and complementary to the information from the Sanger database.We used the data from our cell lines to confirm these findings for four oncogenes . We foun2 value≥0.95, % of subcloning of 48 mutant lines  and RFLP analyses of 38 mutant lines  also showed good concordance with electropherogram measurements  from the SNP array data as described in KRAS, BRAF, and EGFR mutations were mutually exclusive across different tumor types while some PIK3CA mutant cases also harbored one of the other three mutations (Details of the gene mutations and CNGs in the cell lines (n = 114) and tumors (n = 521) are provided in utations  and S2 aKRAS were mutations, with occasional CNGs or both. For EGFR, CNGs were the most frequent alteration, although mutations or both changes were present in prominent subpopulations. For BRAF, mutations and CNGs showed nearly equal frequencies, while both changes were rare. For PIK3CA, CNGs without mutations were the most frequent change  each gene demonstrated a distinct pattern. Most of the alterations in t change .% (as determined by electropherogram mesurement) and CNGs (as determined by qPCR) for the mutant genes in 68 mutant lines including seven lines with double mutations are shown in EGFR.The relationships between of mAKRAS and two mEGFR lines using SNP arrays, and compared the results with MASI status determined by gene specific assays (mA% by direct sequencing and copy number by qPCR). Examples of these comparisons are shown in KRAS lines as one balanced type, one having MASI with CNG and five having UPD. Of the mEGFR lines, one had MASI with CNG and one had MASI with borderline CNG. Of note, the results detected by SNP array were completely concordant with those of the gene specific assays.We evaluated MASI status in seven mFor our studies, determination of relative ratio between mA and wA (mA%) of tumor samples (in contrast to cell lines) requires SNP array analyses. As shown in KRAS  or EGFR  mutations. We determined allelic imbalance (AI) and CNGs of KRAS and EGFR genes using SNP data. The percentage of tumor cell DNA in the samples was determined as described previously and we used appropriately adjusted copy numbers for further analyses. Because MASI frequencies in tumors (as determined by SNP assays) and cell lines (as determined by direct sequencing combined with qPCR) were similar  and KRAS (58%) and lower for BRAF (38%) and PIK3CA (8%). The major type of MASI also showed gene variation  and EGFR (EGFR AI was significantly more frequent in mEGFR (71%) than wild type EGFR cases . By contrast, AI of KRAS was equally observed in mKRAS (62%) and wild type KRAS  and UPD (50%), all KRAS AIs in wild type KRAS cases were caused by UPD.We determined AI in both wild type and mutant case for  18, 40%). HoweverRAS 63%, . While EEGFR and PIK3CA) were present in four cell lines  and these two mutations have been described as usually or always being in cisSecond site (double) mutations in the same gene  showed over 90% of mA%.Subrenal capsule mice xenografts were directly established from primary human NSCLCs. These samples have the following advantage: 1) less manipulation than cell lines , and 2) lack of human normal stromal contamination  samples . We confTo investigate whether CNGs were reflected in increased transcriptional activity, we compared mRNA expression with copy number for 70 mutant cell lines (with or without MASI). As shown in We then investigated whether the increased mA% of MASI lines were maintained after transcription. Using a subset of 35 mutant cell lines (with or without MASI), we compared the mA% of genomic DNA with the values from cDNA . There w2 = 0.97, data not shown). HBEC cultures and wild type tumor cell lines had comparably low levels of activity (KRAS CNGs (without mutation) and those with balanced mutations (without CNGs) had significant 11–12 fold increases in GTPase activity. Cell lines having UPD (without CNGs) had a modest (approximately 50%) increase compared to the balanced mutant lines, although this increase was not significant. However mutant lines having MASI with CNGs had a significantly increased mean activity when compared to the other mutant groups.We evaluated ras GTPase activity by ELISA for 36 cell lines including 26 lung, five colorectal, three pancreatic cancer lines and two HBEC lines . The linactivity . Both liEGFR mutant lines were sensitive at a clinically achievable concentration (<1 µM). We correlated these data with the presence or absence of MASI  and Asian (n = 161) populations obtained from five different institutions and correlated the data with clinical and other findings  and 29 KRAS CNGs (10%). As demonstrated previously in KRAS CNGs were more frequent in mKRAS than in wild type tumors. Because KRAS CNGs were closely associated with mKRAS, KRAS CNGs demonstrated similar associations as have been previously described for mKRAS . Gender differences were not significant for either mutations or CNGs.We determined the mutational status and copy numbers of findings . EGFR muKRAS alterations on clinical outcome of 237 resected lung adenocarcinoma tumors which were limited to stage I–III cases with survival data. Patients with mKRAS tumors (P = 0.2) or KRAS CNGs (P = 0.1) alone had a trend to be associated with poor prognosis. Tumors having both alterations, while present in a small subpopulation (n = 6), had worse prognosis of borderline significance . We found, as expected, that most inactivating mutations of tumor suppressor genes were frequently accompanied by loss of the wild type allele. However, our earlier observations on homozygosity of oncogenes were confirmed by the finding that 20% of five activating oncogene mutations were homozygous in cell lines derived from multiple tumor types. As discussed below, the true incidence of MASI is considerably higher as quatitative copy number data are missing in the Sanger database. Thus MASI, while a long observed and expected phenomenon for tumor suppressor genes, is also present in an important subset of cells harboring mutant oncogenes. Other published evidence supports this concept Detection of MASI of an oncogene requires three basic determinations: 1) detection of an oncogenic mutation; 2) copy number enumeration of the mutant gene in the tumor cells and 3) determination of the relative ratio of the mutant:wild type allele (mA%). Standard and widely accepted methods for the first two determinations exist including direct sequencing for mutations, and qPCR, FISH, aCGH or SNP analyses for CNGs KRAS and EGFR genes. The incidences of MASI in lung cancer cell lines and tumors for these two genes were not significantly different. However, the incidences of MASI for individual oncogenes showed differences, with high frequencies for EGFR and KRAS, intermediate for BRAF and low for PIK3CA. These differences may reflect variations in the oncogenic potential of the individual gene mutations. The frequencies of the two major forms of MASI also demonstrated individual gene differences. For EGFR and BRAF, the most frequent type was MASI with CNGs, while for KRAS, the frequencies of MASI with CNGs and UPD were similar.Four types of inter-relationships between mA and wA were found: a) balanced type, with mutant:wild type allele ratio of approximately one (MASI not present); b) MASI  with CNG; c) uniparental disomy (complete MASI without CNG); and d) reverse MASI . For 75 mutations (in four genes) present in 68 cell lines the overall incidence of MASI was 48%, while only a single example of reverse MASI was identified (p<0.0001). Thus allelic imbalance almost invariably targets the mutant allele. Our previous observations regarding allelic imbalance (obtained by a variety of techniques including subcloning) are consistent with our present findings KRAS and EGFR genes and CNGs of the EGFR gene are well described KRAS CNGs in human tumors is sparse KRAS CNGs were relatively frequent. Of interest, KRAS CNGs showed the same clinico-pathological associations as those previously described for KRAS mutations – relationship to smoking status, non-Asian ethnicity and mutual exclusivity with EGFR mutations While mutations of the JAK2EGFR pathway genes, especially for KRAS gene . Relatively little data exists in the literature for KRAS CNGs in human tumors. Furthermore, KRAS homozygosity was observed independent of mutational status as previously described KRAS can also inhibit lung carcinogenesis in mice While inherited UPD is associated with developmental disorders. the role of acquired UPD in cancer development is poorly understood KRAS mutations did not exert an equal effect on tumor cells KRAS mutations could be broadly divided into KRAS-dependent and KRAS-independent groups. The vast majority of KRAS-dependent lines exhibited focal KRAS CNGs, in contrast to KRAS-independent lines. This study provides further evidence that the combination of KRAS mutations and CNGs act synergistically. Our previous findings that EGFR mutations were associated with tumor initiation while EGFR CNG might be more regarded as a tumor progression event, provide further evidence of their co-operative role in tumorgenesis MASI has apparent biological and clinical significance. MASI at the genomic level was precisely maintained after transcription. While mutations, CNGs and allelic imbalance of mA and wA may all contribute to tumorigenesis, combinations of the three events may be more effective than any single event. Evidence for this concept was provided by our finding that the combination of mutation and CNGs acted synergistically to enhance ras GTPase activity. A recent report found that all EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, copy number gains and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.MASI in its various forms is frequently present in mutant Table S1a) Mutant cell lines of KRAS, EGFR, BRAF, and/or PIK3CA genes (n = 68) mA%, mutant allele proportion (%); *, cell line with both KRAS and PIK3CA mutations; **, cell line with both BRAF and PIK3CA mutations; ***, blanked values are mA% of second mutations of same gene (D549N for PIK3CA and T790M for EGFR). b) Wild type cell lines of KRAS, EGFR, BRAF, and PIK3CA genes (n = 46)(0.04 MB XLS)Click here for additional data file.Table S2a) Summary of 288 lung adenocarcinomas from five institutes b) Summary of 45 lung adenocarcinomas with SNP array data c) The association between KRAS and EGFR alterations and clinicopathological factors in 45 lung adenocarcinomas with SNP *, P value was calculated between Gain and Neutral; **, P value was calculated between Never smoker and Ever smoker. d) Summary of 60 colorectal cancer tumors(0.17 MB XLS)Click here for additional data file.Table S3a) Primer sequences for DNA sequencing b) Primer sequences for cDNA sequencing *, These primers were also used to detect KRAS or EGFR mutations in subrenal capsule mice xenografts of primary human NSCLCs because these primers are specific for human origin and no PCR product are amplified from mouse cDNA as PCR template. c) Primer sequences for restriction fragment length polymorphism *, The substitution of third letter in KRAS codon 61 (limited to CAT or CAC mutation) can change representative amino acid (Gltamine to Histysine). d) Primer sequences for copy number analyses by quantitative PCR (qPCR) assay e) Relative mRNA expression analyses by qPCR(0.03 MB XLS)Click here for additional data file.Table S4The accuracy of proportion of mutant allele (mA%) of direct sequencing was evaluated by 14 kinds of plasmids mixture experiment. We mixed mutant plasmid with corresponding wild type plasmid at various ratios (5 to 7 points) and amplified the mixed plasmid as a template of PCR. PCR products were directly sequenced and the mA% were determined by measurement of sequeincing electropherograms. Finally, we confirmed the linearity between the actual mixed proportion of mutant and wild type plasmids and mA% detected by direct sequencing. The results of the sequencing method were highly concordant with the actual mixture percentage of mutant and wild type plasmids in all 24 trend lines for four genes tested .(0.02 MB XLS)Click here for additional data file.Table S5CNG, copy number gain; Both, cases with both mutations and CNGs; NS, not significant (P>0.1); *, 314 tumors were analyzed because of lack of mutational and copy number data of EGFR gene in 19 Estonia cases; **, data were combined current study and our previous studies - Yamamoto et al (Cancer Res 68: 6913–6921) and Gandhi et al (PLoS ONE 4: e4576).(0.02 MB XLS)Click here for additional data file.Table S6CRC, colorectal cancer; PAC, pancreatic cancer; MASI, mutant allele specific imbalance; UPD, uniparental disomy; CNG, copy number gain; *, limited to 45 lung adenocarcinomas with SNP data; **, because SNP array can not distinguish between MASI and reverse MASI and because incidence of reverse MASI in cell lines is low, we defined tumors harboring allelic imbalance with CNG as MASI with CNG.(0.02 MB XLS)Click here for additional data file.Table S7All other 35 cell lines tested (except for 3 EGFR or HER2 copy number gain cell lines) were resistant for gefitinib (IC50>10 mM)(0.03 MB XLS)Click here for additional data file.Figure S1Calculation method of mutant allele proportion (mA%) for deletion (or insertion) type of mutations is shown. The average of mA% of the first five different waves from the beginning of mutations is calculated.(0.42 MB PPT)Click here for additional data file.Figure S2We performed restriction fragment length polymorphism (RFLP) method to quantify mutant allele . Examples for two types of mutations (KRAS codon 12 mutations and EGFR exon 19 deletion type mutations) are shown. Percent of mutant allele (%mA) detected by measurement of sequencing electropherogram has good concordance with %mA detected by subclonig and RFLP methods .(0.59 MB PPT)Click here for additional data file.Figure S3Mutant allele specific imbalance (MASI) can be observed in mice xenograft samples. Complete MASI is present in xenogragts established from patients with stage Ib to IIIa.(0.16 MB PPT)Click here for additional data file.Figure S4Ras GTPase activity in 36 cell lines is shown. MASI, mutant allele specific imbalance; WT, wild type; CNG, copy number gain; HBEC, human bronchial epithelial cell; The prefix m- means mutant.(0.18 MB PPT)Click here for additional data file.


Zea mays that are robust enough to use with other closely related species.Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis) supported this assumption by successfully producing single amplicons for each primer pair.Primers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR) as visualized on agarose gels and subsequently verified by q2(RT)PCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico and in vitro data are presented to support this assumption.Due to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RT)PCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both  Chloroplasts are semi-autonomous organelles believed to have developed from free-living photosynthetic bacteria ,2 They aPoaceae) also offer unique opportunities to study differential plastid gene expression. The presence of closely related species with either C3 or C4 photosynthetic capabilities enables comparison of dimorphic C4 chloroplast development to monomorphic C3 species. In addition, grasses such as switchgrass (Panicum virgatum) and miscanthus (Miscanthus sinensis) are the subjects of an increased focus on cellulosic ethanol production , NAPD-Malic enzyme accession number [GenBank: J05130], and NAPD-Malate dehydrogenase accession number [GenBank: AY105634] [q2(RT)PCR primer design and validation procedural flow can be seen in Figure Y105634] . Gene seY105634] . SuggestY105634]  and editY105634]  for ampl® according to the manufacturer's suggested protocol, Invitrogen . Each extraction was checked for a DNA/RNA concentration utilizing a Spectronic® Genesys™ spectrophotometer . Ambion's Turbo DNase-free™ or DNase-free™,  was used according to manufacturers suggested protocol to remove contaminating DNA. DNase treatments were repeated until each RNA extraction was visually confirmed to be DNA free by end point PCR reactions.Maize (inbred line T42) stage 2 leaves were dissected into basal, mid, and tip sectional tissue as described in Cahoon, et al  and mixe®Green utilizing approximately 200 ng/ul cleaned RNA per reaction for each of the three tissue samples. Amplicon presence and purity was confirmed by analysis of the IQ™5 Optical System's melt curve, melt curve peak, and PCR amplification software.Each gene primer pair was verified utilizing BioRad's  iCycler and IQ™5 Multi-color Real-Time PCR Detection System. 25 μl reactions were prepared with BioRad iScript™ One-Step RT-PCR kit with SYBERRT-PCR: reverse transcriptase PCR; q2(RT)PCR: quantitative real time reverse transcriptase PCRThe authors declare that they have no competing interests.RMS designed the primers, performed the q2(RT)PCR reactions, interpreted reaction results and prepared the manuscript, SND processed and performed the q2(RT)PCR for the majority of the switchgrass samples, ABC was involved in the conception and design of the project, revised and edited the manuscript, and is the PI of the laboratory.q2(RT)PCR primer pairs with their corresponding genes, category, and optimal elongation time, and annealing temperatures. All primers were initially designed to produce amplicons between 75 and 150 base pairs from specific maize RNAs. Primer pair "category" is based on the specificity of the reaction conditions required to produce a single amplicon. Category 1 pairs produce a single product within a range of conditions, category 2 pairs require more stringent annealing or elongation times, and category 3 are genes with intercellular homologs (see text for full descriptions).Click here for filein silico comparison of maize primers to other plastome sequences. Primer sequences were compared to representative plastomes from the BEP and PACCAD clades, as well as Arabidopsis. Success was gauged by overall percent homology and the presence of mismatched bases at the 3' end of the primers. These data are summarized in Figure Click here for file


Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the  Ebola virus is among the most lethal microbes known to man, with case fatality rates often exceeding 80%. Since its discovery in 1976, outbreaks have been sporadic and geographically restricted, primarily to areas of Central Africa. However, concern about the natural or unnatural introduction of Ebola outside of the endemic areas has dramatically increased both research interest and public awareness. A number of candidate vaccines have been developed to combat Ebola virus, and these vaccines have shown varying degrees of success in nonhuman primate models. Safety is a significant concern for any vaccine and in particular for vaccines that replicate in the host. Here, we evaluated the safety of our replication-competent vesicular stomatitus virus (VSV)-based Ebola vaccine in SHIV-infected rhesus monkeys. We found that the vaccine caused no evidence of overt illness in any of these immunocompromised animals. We also demonstrated that this vaccine partially protected the SHIV-infected monkeys against a lethal Ebola challenge and that there appears to be an association with levels of CD4+ lymphocytes and survival. Our study suggests that the VSV-based Ebola vaccine will be safe in immunocompromised populations and supports further study and development of this promising vaccine platform for its use in humans. Sudan ebolavirus (SEBOV) to up to 90% for Zaire ebolavirus (ZEBOV) (reviewed in Ebola virus (EBOV) has been associated with sporadic episodes of hemorrhagic fever (HF) that produce severe disease in infected patients. Mortality rates in outbreaks have ranged from 50% for While there are no vaccines or postexposure treatment modalities available for preventing or managing EBOV infections there are at least four different vaccine systems that have shown promise in completely protecting nonhuman primates against a lethal EBOV challenge Of these two leading EBOV vaccine candidates that can confer protection as single injection vaccines each has advantages and disadvantages. Adenovirus vectors are highly immunogenic as documented by clinical trials evaluating gene transfer efficacy and immune responses. Because they are replication-defective adenovirus vectors are also perceived to be safer for human use than a replication-competent vaccine. The most significant challenge for the adenovirus-based vaccines is the concern that a significant portion of the global population has pre-existing antibodies against the adenovirus vector which may affect efficacy Because EBOV outbreaks in man have occurred exclusively in Central and Western Africa, the populations in this region are among those that may benefit from the development and availability of an EBOV vaccine. However, populations in this region are among the most medically disadvantaged in the world. In particular, the prevalence of individuals with a compromised immune system is high and HIV infections rates range up to 10% or more in this area The recombinant VSV expressing the glycoprotein (GP) of ZEBOV (strain Mayinga) (VSVΔG/ZEBOVGP) was generated as described recently using the infectious clone for the VSV, Indiana serotype Macaca mulatta) (5–10 kg) were used for these studies. The macaques were infected three months prior to the current study with SHIV162p3 . These animals all had clinical laboratory evidence of SHIV infection as evidenced by reduced CD4+ T cell counts, decreased ratios of CD4+/CD8+ T cells . The white blood cell differentials were performed manually on Wright-stained blood smears. Serum samples were tested for concentrations of albumin (ALB), amylase (AMY), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), glucose (GLU), cholesterol (CHOL), total protein (TP), total bilirubin (TBIL), blood urea nitrogen (BUN), and creatinine (CRE) by using a Piccolo Point-Of-Care Blood Analyzer .100 ul of whole blood was added to a 12×75 tube and incubate with the antibodies for 15 minutes at room temperature. The samples was then lysed and fixed in 1% paraformaldehyde and washed three times in PBS. Samples were analyzed on a Becton Dickinson FACS Calibur . All antibodies were purchased from Becton Dickinson; clones used were CD3 – SP34, CD4 – L200, CD8 – RPA-T8 and CD20 – 2H7.5′ AGTATGGGCAGCAAATGAAT 3′ (forward primer), SIV-R 5′TTC TCTTCTGCGTG AATGC 3′ (reverse primer), SIV-P 6FAM-AGATTTGGATTAGCAGAAAGCCTGTTG GA-TAMRA (TaqMan probe) in a 7700 Sequence Detection System . The signal was then compared to a standard curve of known concentrations to determine the viral copies present in each sample. The assay lower limit was 40 copies/ml.For measurement of plasma SIV RNA levels, a quantitative TaqMan RNA reverse transcription-PCR (RT-PCR) assay  was used, which targets a conserved region of SIV gag and has an accurate detection limit as low as 200 RNA copies/ml. Briefly, isolated plasma viral RNA was used to generate cDNA using One-Step RT-PCR Master Mix (Applied Biosystems). The samples were then amplified as previously described RNA was isolated from blood and swabs using Tripure Reagent . For the detection of VSV we used a Q-RT-PCR assay targeting the matrix gene . ZEBOV RNA was detected using a Q-RT-PCR assay targeting the L gene . The low detection limit for this ZEBOV assay is 0.1 pfu/ml of plasma. Virus titration was performed by plaque assay on Vero E6 cells from all blood and selected organ  and swab samples. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero E6 monolayers in duplicate wells (0.2 ml per well); thus, the limit for detection was 25 pfu/ml.IgG antibodies against ZEBOV were detected with an Enzyme-Linked Immunosorbent Assay (ELISA) using purified virus particles as an antigen source as previously described Necropsies were performed on each animal and selected tissues were collected for histological analysis. Histology and immunohistochemistry were performed as previously described for ZEBOV-infected monkeys 3 pfu/ml) was detected only on day 2 after vaccination by virus isolation  and the remaining three animals (Controls #1–3) received sterile saline. The animals were monitored closely for clinical symptoms and shedding of recombinant VSVs. None of the animals vaccinated with VSVΔG/ZEBOVGP or treated with saline showed overt fever or any evidence of clinical illness during the 31 day vaccination period. Importantly, no evidence of reaction at the vaccine injection site was noted among any of the VSVΔG/ZEBOVGP-vaccinated animals nor was any change noted in activity or behavior during the vaccination phase of the study (day 0 to day 31 after vaccination). In addition, no changes were detected in hematology or clinical chemistry following vaccination. A mild VSVΔG/ZEBOVGP viremia . Four of the six VSVΔG/ZEBOVGP-vaccinated SHIV-infected monkeys and both of the placebo control animals started to show clinical signs of disease on day 6 after challenge including fever  and lymphopenia and thrombocytopenia  . Disease4.5 log pfu/ml) (2 log pfu/ml) (4.2 log pfu/ml). RT-PCR was more sensitive and showed evidence of ZEBOV in plasma of this animal (Subject #5) at day 6. In addition, RT-PCR was more sensitive in detecting ZEBOV in swabs which were positive on a number of samples derived from Subject #5 at day 6 and day 10  . In comp pfu/ml) . ZEBOV wd day 10 . In contThe four surviving VSVΔG/ZEBOVGP-vaccinated macaques  were euthanized 28 days after the ZEBOV challenge to perform a virological and pathological examination of tissues. Organ infectivity titration from these four animals showed no evidence of ZEBOV in any of the tissues examined. In comparison, ZEBOV was recovered from tissues of both VSVΔG/ZEBOVGP-vaccinated animals that succumbed  and both SHIV-infected control animals. Organ titers of infectious ZEBOV were consistent with values previously reported for immunocompetent ZEBOV-infected rhesus macaques Pathological and immunohistochemical evaluation of tissues from the four VSVΔG/ZEBOVGP-vaccinated animals  that survived ZEBOV challenge showed no evidence of ZEBOV antigen. In contrast, ZEBOV antigen was readily detected in typical target organs  of the two VSVΔG/ZEBOVGP-vaccinated animals that succumbed to ZEBOV challenge   and the While cellular immune responses against ZEBOV GP in macaques vaccinated with VSVΔG/ZEBOVGP vectors have been difficult to detect before challenge in previous studies 0–2×105 pfu (5 pfu of the VSV-based ZEBOV vaccine (VSVΔG/ZEBOVGP) resulted in no clinical symptoms An often raised concern regarding the use of the recombinant VSV vaccine platform in humans is related to the fact that this is a replication-competent vaccine, and thus demonstration of safety is of paramount importance. Taking into account our previous work it is not surprising that the VSVΔG/ZEBOVGP was tolerated well in our SHIV-infected macaques. Specifically, we failed to observe evidence of any adverse events in a large cohort of over 90 macaques receiving VSV vectors expressing different GPs from viral HF agents   did not cause any illness in immunocompromised SHIV-infected rhesus macaques and resulted in sufficient protective efficacy in all but the most severely compromised animals against a lethal ZEBOV challenge. Protection in the immunocompromised macaques appeared to be dependent on CD4+ T cells rather than the development of EBOV-specific antibodies. This provides strong support for the safety of the VSV-based vectors and further development of this promising vaccine platform for its use in humans. While these data are very encouraging, as the number of SHIV-infected macaques in the current study was small, additional safety studies will be needed in order to determine whether vaccines based on attenuated VSV will ultimately prove safe in immunocompromised humans.


Light chain amyloidosis (AL) is a rare plasma cell dyscrasia associated with poor survival especially in the setting of heart failure. Late gadolinium enhancement (LGE) on cardiac MRI was recently found to correlate with myocardial amyloid deposition but the prognostic role is not established. The aim is to determine the prognostic significance of LGE in AL by comparing long term survival of AL patients with and without LGE.Twenty nine consecutive patients  with biopsy-proven AL undergoing cardiac MRI with gadolinium as part of AL workup were included. Survival was prospectively followed 29 months (median) following MRI and compared between those with and without LGE by Kaplan-Meier and log-rank analyses.LGE was positive in 23 subjects (79%) and negative in 6 (21%). Left ventricular ejection fraction was 66 ± 17% in LGE-positive and 69 ± 12% in LGE-negative patients (p = 0.8). Overall 1-year mortality was 36%. On follow-up, 14/23 LGE-positive and none of LGE-negative patients died (log rank p = 0.0061). Presenting New York Heart Association heart failure class was also associated with poor survival (p = 0.0059). Survival between two LGE groups stratified by heart failure class still showed a significant difference by a stratified log-rank test (p = 0.04).Late gadolinium enhancement is common and is associated with poor long-term survival in light chain amyloidosis, even after adjustment for heart failure class presentation. The prognostic significance of late gadolinium enhancement in this disease may be useful in patient risk-stratification. Light chain or primary amyloidosis (AL) is a plasma cell dyscrasia with production and abnormal deposition of insoluble fibrillar proteins derived from immunoglobulin light chains ,2. It isAmong 36 consecutive patients with biopsy-proven diagnoses of light chain amyloidosis seen by our Oncology or Cardiology Division from April 2005–November 2008, 29 underwent cardiac MRI as part of routine workup and were included in this study. Seven patients did not undergo cardiac MRI because of low glomerular filtration rates unsuitable for gadolinium administration and were not included in this study. The study was approved by the local Institutional Review Board (IRB). Twenty five patients signed informed consent as part of a longitudinal study of light chain amyloidosis and were prospectively followed. Four patients undergoing workup expired before recruitment for the study and waiver of consent was authorized by the IRB for data collection. Their data were included to ensure 100% capture of consecutively evaluated AL subjects in our institution. Clinical data was obtained that included presenting hemodynamic, laboratory profile and New York Heart Association heart failure functional class (I–IV). The latter was adjudicated by a cardiologist based on presenting symptoms and signs according to established clinical standards A General Electric 1.5 Tesla CV scanner was used with 8-channel cardiac coil. Cardiac gated cine segmented steady state free precession pulse sequence was performed on short axis view orthogonal to the long axis of the left ventricle to assess left ventricular function by manual tracing of endocardial borders using ReportCard software . The imaging parameters used were: field of view 34–40 cm, 160 × 256 matrix, 7–8 mm slice thickness, 2–3 mm gap, flip angle 45°, retrospective gating. For late gadolinium enhancement imaging, 0.1 mmol/kg of gadolinium  was injected and imaging started after ~5 minute delay in short axis and multiple long axis views. Cardiac gated segmented inversion-recovery prepared gradient echo pulse sequence was used with field of view 38–42 cm, matrix of 256 × 192–256, slice thickness of 7–8 mm, interslice gap of 2–3 mm, inversion time of 175–300 ms adjusted to null normal myocardial signal, number of excitations of 1–2 and 2 R-R intervals. The optimal inversion time that nulls normal myocardium was determined by acquiring multiple images of the same midventricular view using different inversion times. If a significant portion of the myocardium goes through the null point earlier than the left ventricular cavity blood pool this area was considered to represent myocardial disease and subsequently the inversion time was then adjusted to null the remaining myocardial areas , an approach that has been previously validated with histologic gold standard [Data are expressed as mean ± standard deviation. Continuous variables were compared between groups using Student's t-test  or Mann-Whitney rank sum test  using SigmaStat 3.5 software . Categorical variables were compared using Fisher's exact test. Stratified estimates of survival probabilities were computed using Kaplan-Meier estimators  with logThere were 14 females with mean age of 62 ± 11 years. Biopsy was positive for amyloid in the heart  of patients Figure  but in sFollow-up of survival status was done 26 ± 13 (median 29) months following cardiac MRI. Overall 1-year mortality was 36% (46% for LGE-positive patients). The immediate cause of mortality was cardiac-related in the majority of patients (Table The study's novel observation is that the presence of late gadolinium enhancement on cardiac MRI is associated with poor survival on long-term follow up in light chain amyloidosis patients and that this MRI finding had independent and additive value to clinical assessment of heart failure status. This may be useful in identifying high risk patients and adds prognostic value to the known diagnostic value of LGE in light chain amyloidosis.Light chain amyloidosis is a rare condition but cardiac involvement is important. Median survival following standard chemotherapy was reported to be 12–18 months but with heart failure, the survival is reduced to 4 months . Light cLate gadolinium enhancement and presenting heart failure class were the strongest univariate predictors of mortality in our cohort. Heart failure is known to be associated with poor outcomes in these patients ,5. StratThe mechanism underlying adverse outcome in patients with late gadolinium enhancement, a marker of cardiac amyloid involvement, is not established but may lie in tissue toxicity from light chain amyloid deposition. Autopsy studies reveal that the presence of amyloid in the perivascular and interstitial space is associated with histologic evidence of myocardial ischemia in 74% of patients . EpicardIn conclusion, late gadolinium enhancement on cardiac magnetic resonance imaging is associated with poor long-term survival in light chain AL amyloidosis, even adjusted for heart failure class presentation. This finding may be useful in identifying high risk patients and adds prognostic value to the known diagnostic value of LGE in light chain amyloidosis.The authors declare that they have no competing interests.RQM was involved in conception and design, subject enrollment, acquisition of data, analysis and interpretation of data, drafting and final approval of the manuscript. RC was involved in acquisition of data, interpretation of data, revision and final approval of the manuscript. AS was involved in statistical analysis and interpretation of data, revision and final approval of the manuscript. MB and ST were involved in subject enrollment, revision and final approval of the manuscript. PH was involved in conception and design, subject enrollment, acquisition and interpretation of data, revision and final approval of the manuscript.The pre-publication history for this paper can be accessed here:


Each group was exposed to purified highly pathogenic AIV H7N3 strain at a dose 105 embryo lethal dose (ELD50). We observed that nucleoprotein (NP) antiserum significantly protected birds from viral infection induced morbidity, mortality and lowered viral shedding compared with antiserum from individual viral proteins or mixed polypeptides/proteins inclusive of NP component. The capability of individual viral polypeptide specific antisera to protect against viral challenges in decreasing order was nucleoprotein (NP) > hemagglutinin (HA) > neuraminidase (NA) > viral proteins mix > viral polymerase (PM) > non-structural proteins (NS). Our data provide proof of concept for potential utilization of passive immunization in protecting poultry industry during infection outbreaks. Furthermore conserved nature of avian NP makes it an ideal candidate to produce antiserum protective against viral infection.Our studies were aimed at developing a vaccination strategy that could provide protection against highly pathogenic avian influenza virus (AIV), H7N3 or its variants outbreaks. A purified viral stock of highly pathogenic H7N3 isolate was lysed to isolate viral proteins by electrophresing on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by their elution from gel through trituration in phosphate buffered saline (PBS). Overall, five isolated viral polypeptides/proteins upon characterization were used to prepare hyperimmune monovalent serum against respective polypeptides independently and a mixture of all five in poultry birds, and specificity confirmation of each antiserum through dot blot and Western blotting. Antiserum generated from various group birds was pooled and evaluated in 2-week old broiler chicken, for its protection against viral challenge. To evaluate  Avian influenza virus (AIV) besides reducing commercial production of poultry is also a causative agent for influenza among humans by cross-species infections . The virAvian influenza (AI) has emerged as a disease with significant potential to disrupt commercial poultry production, resulting in heavy losses to poultry farmers in several parts of the world. Due to fastidious viral genome, conventional antivirals against AIV are unable to control the infection and very few effective vaccines are available. Moreover, geographic strain variations have made it difficult to implement universal avian influenza vaccine strategy. As such, there has been an urgent need to develop broad spectrum antivirals against AIV or vaccines capable of coping with viral genomic changes. One of the most plausible options to control AI is development of regional immunization programs against the serotype involved in an outbreak. However, as the immunization has to be carried out prior to disease for establishing therapeutic levels of antibodies against the infection, in case of its sudden outbreak such control measures are not possible. Passive immunization has emerged as an effective therapeutic tool in the face of an outbreak; however its effectiveness in the case of AIV has not yet been investigated. During past decade, AIV, H7 serotype has caused high poultry birds mortality in different countries including Pakistan . The whoOur initial work on SDS-PAGE analysis of H7N3 viral lysate showed five major viral proteins: high molecular weight polymerase (PM), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA) and non-structural protein (NS) as shown in Figure th all the birds in PM, NS and control  were dead. Mortality was associated with extensive morbidity in polypeptides groups showing less protection. One of the groups was vaccinated with antiserum generated from a mixture of all five peptides . It was intriguing to note that on day 4th two birds died in this group without any further mortality thus showing 80% protection. No mortality (100% protection) was observed in birds pre-vaccinated with hemagglutinin, nucleoprotein, and neuraminidase antisera. However, morbidity and viral shedding revealed 80–100% birds infected in HA vaccinated group, 20 – 60% in NP and 80–100% in NA groups . Birds' morbidity, mortality and cloacal shedding were observed over a time period of two-week. Four out of the six vaccinated group showed protection from lethal viral challenge whereas negative control group showed highest level of mortality, morbidity and cloacal shedding. The level of protection in the four groups varied and nucleoprotein antiserum vaccinated group birds showed highest protection revealed by least mortality, and low viral shedding (60%). The birds passively immunized with polymerase and non-structural protein antiserum showed no protection at all. Upon viral challenge, seven out of ten birds died in polymerase and non-structural protein antiserum vaccinated groups, whereas eight in non-vaccinated control group Table . This trth. The nucleoprotein antiserum immunized group showed the least morbidity (maximum 60%) at day 4 along-with no mortality (0%) and lowest level of cloacal shedding makes it a potential candidature for poultry vaccine against H7N3 especially through passive immunization route.Morbidity describes disease condition and prevalence of various symptoms associated with viral infection in birds. In case of bird flu outbreak, the infected birds manifest quite distinctive symptoms like ruffled feathers, excessive thirst, areas of diffuse hemorrhage between the hocks and feet, edema surrounding the eyes, watery green diarrhea progressing to white and several others. Mortality in the control (non-vaccinated) and two of the viral peptides  antisera manifesting least protection (0%) was associated with several disease symptoms an indicator for high morbidity (100%). In comparing the data of all protective antisera groups, the level of morbidity was higher in viral polypeptides and neuraminidase groups (100%) followed by hemagglutinin (80%) on day 4In vaccinated groups challenged with lethal AIV, NP groups showed least cloacal shedding of virus among all the groups. All other vaccinated and non-vaccinated control manifested cloacal shedding of virus. These data are quite interesting and will help us in designing future vaccine for AIV in poultry.Infections associated with AIV are threatening economy of several countries throughout the World. Particularly in South-East Asia viral infection has inflicted major losses to poor poultry farm holders as well as it poses a threat of cross-species infection among humans. AIV is a member of Type A group viruses and compared with its counterparts Type B and C has broad host range capable of causing infections in several birds and mammals. One of the major threats of AIV has been its capability to cross-species jumping i.e. from birds to humans .According to a report from the International Federation for Animal Health (IFAH) vaccination strategies for controlling AIV infection in birds is one of the major viable options compared with other control measures . SeveralPassive immunization with antiserum generated from viral polypeptides antigenic determinants has shown significant protection in mammals ,14 and aFour out of six vaccines have given protection in decreasing order NP>HA>NA>viral polypeptides mix. In case of HA, NA and viral polypeptides mix, the level of infection increased from day 0 to day 4 and then it decreased till the end of experiment i.e. day 14. NP antiserum besides providing 100% protection also boosted chick's immunity manifested as sustained resistance against infection  as compared to other vaccine groups. These data suggest that passively transfused anti-NP antibodies have a better antiviral neutralizing effect and overall protection from AIV. Overall, a better protection provided during days 7–14 is due to immune regulation.Considering the situation of developing nations like Pakistan passive immunization strategy will be economical and targeted. Avian Influenza is capable of changing antigenic determinants that leads to inefficacy of vaccines. A locally produced economical vaccine will provide effective and long lasting solution to this pandemic especially the non-variant parts (nucleoproteins) that hold the promising future of AIV vaccines.Prior to beginning this study the protocol was reviewed and approved by the animal biosafety committee of the Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, and all the viral challenges and preparations were conducted at the biosecure facilities of the National Reference Laboratory for Poultry Diseases (NRLPD) at the Animal Sciences Institute, National Agriculture Research Center (NARC), Islamabad, Pakistan.50) titer of the fresh viral stock was determined by classical Reed and Muench [1 to 1012) followed by injecting 0.2 ml of each dilution into the chorioallantoic region of embryonated eggs. The mortality of eggs is recorded and ELD50 calculated as described previously[A previously isolated highly pathogenic AIV serotype H7N3 A/Chicken/Pakistan/Murree/NARC/69/04 (H7N3)  was obtad Muench  methodolreviously.Purified fresh stock of H7N3 AIV was lysed with 4% Triton X-100 using 0.01 M Tris buffer (pH 7.2) in the presence of 1 mM KCl. Viral lysate was stirred for 45 minutes at room temperature followed by centrifugation at 10,000 × g to get the supernatant containing HA, NA and matrix (M) proteins. The pellet containing NP protein was washed with phosphate buffer saline (PBS), by re-centrifuging at 10,000 × g for 1 hour at 4°C. To remove viral DNA/viral particles the supernatant was centrifuged at 200,000 × g by using Beckman ultracentrifuge L8-80 on 50 Ti rotor  for 1 hour to remove the viral DNA and viral particles. The supernatant was collected and dialyzed against 0.01 M PBS for 48 hours. It was again centrifuged at 10,000 × g for 10 minutes to separate M protein out of these preparations and the resulting pellet was suspended in PBS. The supernatant containing HA, NA, polymerase (PM) and non-structural (NS) proteins was collected by centrifuging three times repeatedly at 10,000 × g for 10 minutes at 4°C. The supernatants were dialyzed and the resultant collections were analyzed on 12% polyacrylamide gel. Five bands of AIV proteins separated on the gel were cut, triturated and diluted with 1 ml of normal saline solution (NSS). The material was centrifuged at 1000 × g for 10 min and supernatant was quantified by Lowry's method . Each poDot-ELISA was standardized and performed to check the specificity of each polypeptide specific antisera against AI H7N3 virus. Antigen dots were used in different dilutions ranging from Neat virus to 1:4 dilutions with NSS along with a dot containing BSA as a negative control.5 ELD50. The birds were examined for clinical signs, mortality and cloacal shedding, up to 14 days post-challenge (PC).Broiler chicks tested negative for AIV were divided equally into seven group of ten each. These birds were reared under strict isolation and high security conditions in chicken isolators. At the age of two weeks, birds were passively immunized with 4 ml each of the polypeptide specific antisera. Birds were challenged while rearing in chicken isolators at 24 hours post inoculation (PI) with live virus of AI serotype H7N3 at a dose 10


An accurate, early diagnosis and treatment of adenomatous polyp can curtail progression to colorectal cancer. F-18 fluorodeoxyglucose positron emission tomography (F-18 FDG PET) reveals the biochemical changes associated with the development of many cancers which precede the appearance of gross anatomical changes that may be visualized during surgical resection or via imaging with MR or CT.We detail the history of a 64 year old female who had a whole-body FDG PET scan as a part of an employee wellness program. A dose of 12.2 mCi of F-18 labeled FDG was administered.A focal cecal uptake with a standardized uptake value (SUV) of 8.9 was found on the PET scan. Conversely, only normal mucosa was observed during a colonoscopy done 2 months after the PET scan. Motivated by the PET scan finding, the colonoscopist performed a biopsy which revealed a villous adenoma without high grade dysplasia. Pathology from tissue extracted during an exploratory laparatomy completed one month later found the lesion to be a villous adenoma with high grade dysplasia.Whole-body FDG PET scan revealed the biochemical metabolic changes in malignancy that preceded the appearance of any gross anatomical abnormality. A positive FDG PET scan indicative of colorectal cancer should be followed up with a colonoscopy and biopsy even in a visibly normal mucosa. We hereby report a case of advanced adenoma which was detected on a whole-body FDG PET scan despite the fact that the mucosa had a normal appearance on follow up colonoscopy. F-18 fluorodeoxyglucose positron emission tomography (F-18 FDG PET) reveals the biochemical changes associated with the development of many cancers which precede the appearance of gross anatomical changes that may be visualized during surgical resection or via imaging with MR or CT.A dose of 12.2 mCi was given to the patient following a 4 hour fasting period. The tracer incorporated for 1 hour prior to image acquisition. A 6 bed position, whole-body PET scan was performed on a Siemens HR+ PET scanner. Each bed position included a 2 minute transmission followed by a 5 minute emission scan. Reconstruction parameters were 2 iterations/8 subsets (OSEM) with a 4 mm smoothing Guassian filterThe patient is a 64 year old female who had a whole body FDG-PET scan in June 2004 as part of an employee wellness program. The scan was negative except for a focal abnormality in the cecal area which had an SUV of 8.9 Figure . The patTwo months after the FDG-PET scan, a colonoscopy was performed. There was no visible abnormality in the cecum Figure  but promPolyp is a small growth of tissue shaped like the head or stalk of a mushroom. They are classified histologically as neoplastic (adenomatous) or non neoplastic . AdenomaIn this educational case report, the patient had a high grade villous adenoma which was discovered due to its increased FDG uptake on a whole-body PET scan but the mucosa looked normal on the colonoscopy. FDG PET scanning detects biochemical changes which precede gross morphological changes in cancerous tissue. Increased cell surface glucose transporter protein 1 to 5 and increased level of hexokinase pathway explain the high FDG uptake in malignancy.Narrow Band imaging and Chromoendoscopy with indigo carmine can increase the yield of conventional colonoscopy. ,4 The reAccording to the current guidelines , since aFDG PET has a high sensitivity in diagnosing premalignant and advanced colonic neoplasia -14. Van Whole-body FDG PET scan revealed the biochemical metabolic changes in malignancy that preceded the appearance of any gross anatomical abnormality. A positive FDG PET scan with a focal uptake in colon should be considered a significant finding indicative of colorectal cancer and follow up colonoscopy and biopsy should be recommended even in a visibly normal mucosa.The author(s) declare that they have no competing interests.BR drafted the manuscript, BR and PS reviewed the literature,RC did the colonoscopy; AB admitted the patient and followed up with the patient,JM and MS were involved with imaging the patient; YD interpreted the pathology report.All authors read and approved the final manuscript.


Transmissible gastroenteritis virus [TGEV]), a virus that our laboratory has been studying for 16 years Coronaviridae family Endemic Balkan nephropathy (EBN), a disease restricted to three Balkan countries , is characterized by a progressive shrinking of the kidneys and, in some cases, tumors in the proximal regions of the urinary tract The EBN-associated virus was isolated from primary kidney cells cultures, grown from fresh renal biopsy specimens of clinically confirmed cases of EBN 6 to 107 PFU/mL. No specific neutralization was observed when polyvalent or monoclonal antibodies that neutralized TGEV or the human coronaviruses OC43 or 229E were used in a standard neutralization assay. Furthermore, we observed no reactivity by immunofluorescence microscopy with the same antisera and specific monoclonal antibodies A titration method was set up for the EBN-associated virus in Vero cells, as described for coronaviruses Since coronavirus morphology is easily recognized by electron microscopy, Vero cells infected with the EBN-associated virus were embedded in resin for electron microscopy, and ultrathin sections were examined. Coronaviruses interacting with the cell membrane or inside the cell cytoplasm were easily seen when grown in swine testicle cells , A and CSupernatants of Vero cells infected with the EBN-associated virus were concentrated 100-fold by ultracentrifugation and visualized by negative staining with 2% uranyl acetate. Using purified TGEV as a standard, we observed only one type of spherical virion with a homogeneous mean virion size 28.4 nm ± 2 nm in diameter  , F. In cThe virus source used in these experiments is the same as that previously analyzed


Newer antiretroviral (ARV) agents have improved pharmacokinetics, potency, and tolerability and have enabled the design of regimens with improved virologic outcomes. Successful antiretroviral therapy is dependent on patient adherence. In previous research, we validated a subset of items from the ACTG adherence battery as prognostic of virologic suppression at 6 months and correlated with adherence estimates from the Medication Event Monitoring System (MEMS). The objective of the current study was to validate the longitudinal use of the Owen Clinic adherence index in analyses of time to initial virologic suppression and maintenance of suppression.score ≥ 5 (95% CI: 0.19–0.69) [reference: <5]. In the final generalized estimating equations (GEE) logistic regression model the adjusted odds ratio for time-updated adherence score was 0.17score ≥ 5 (0.05–0.66) [reference: <5].278 patients  met inclusion criteria. Median [range] time on the first regimen during the study period was 286 (30 – 1221) days. 217 patients (78%) achieved an undetectable plasma viral load (pVL) at median 63 days. 8.3% (18/217) of patients experienced viral rebound (pVL > 400) after initial suppression. Adherence scores varied from 0 – 25 . The lowest detectable adherence score cut point using this instrument was ≥ 5 for both initial suppression and maintenance of suppression. In the final Cox model of time to first undetectable pVL, controlling for prior treatment experience and baseline viral load, the adjusted hazard ratio for time updated adherence score was 0.36A brief, longitudinally administered self report adherence instrument predicted both initial virologic suppression and maintenance of suppression in patients using contemporary ARV regimens. The survey can be used for identification of sub-optimal adherence with subsequent appropriate intervention. In previous research, we validated a subset of items from the ACTG adherence battery as prognostic of virologic suppression at 6 months and moderately correlated with adherence estimates from the Medication Event Monitoring System (MEMS) . The obj10 transformed HIV plasma viral load were 173 and 5.0, respectively. Index antiretroviral regimens were distributed as follows: ≥ 2 nucleoside reverse transcriptase inhibitors (NRTIs) + 1 boosted protease inhibitor (PI/r) 73%, ≥ 2 NRTIs + 1 non-nucleoside reverse transcriptase inhibitor (NNRTI) 23%, and other regimens 4%. Enfuvirtide was included as part of the index regimen in only two patients. Median [IQR] days on the index regimen was 286 [115–566] overall. According to prior antiretroviral experience, the median [IQR] days on therapy was 285 [116–566] for treatment naïve patients and 286 [93–562] for treatment experienced patients. 217 patients (78%) achieved an undetectable pVL at median 63 days. 8.3% (18/217) of patients experienced viral rebound (pVL > 400) after initial suppression. The median number of per-patient administrations of the adherence instrument was 4, varying from 1 to 27 administrations. Adherence scores varied from 0 – 25 .Study eligibility criteria were met by 278 patients whose baseline characteristics are presented in Table Of the 1155 records in the final analysis dataset representing the longitudinal histories of 278 patients, HIV viral load and adherence were measured on the same date in 556 (48%) records. Of the 1155 records, 599 (52%) represented missing adherence scores at dates of viral load measurement. Of the 599 missing adherence scores, 426 were imputed using the last observation carried forward approach (LOCF) and 173 were imputed by backfilling values. Even though these missing adherence scores technically represent missing values at the time the viral load measures were taken, they conceptually represent values that were obtained at a different time point than the viral load measures. These instances typically represent patients for whom blood is drawn either before of after a clinic visit at which adherence assessment was conducted. The median (IQR) time between the regimen start date and date of the first recorded adherence score was 21 (13–60) days.10 HIV plasma viral load, prior antiretroviral treatment experience , index regimen type. Of these potential covariates, baseline HIV viral load and race were significantly (p < 0.05) associated with time to viral suppression. Table Because the distribution of adherence scores was highly skewed Figure  we modelTable In the developmental phase of adherence measurement in our clinic, we constructed a 5-item instrument whose individual items were selected from the 51-item ACTG adherence battery  on the bThese findings are not directly comparable to the effects demonstrated in our earlier study for several reasons including: (1) period effects (1998 – 1999 vs. 2003 – 2006) associated with changes in potency and simplicity of antiretroviral regimens; (2) differences in prior treatment experience ; (3) conditions of adherence measurement (written completion [earlier study] vs. computer assisted [current study]); and (4) differences in analytic approach . Nonetheless, the current results contribute to the predictive validation of the instrument as it has been used in routine clinical care of patients on antiretroviral therapy.In a recent review of the status of HIV adherence measurement, Chesney presented a conceptual model of adherence assessment and intervention, distinguishing research from clinical applications, and resource-rich from resource-poor settings. In discussing the "elusive gold standard" of adherence measurement, she emphasized that "efforts should continue to develop a portfolio of different valid and reliable self-report measures with varying strengths and weaknesses that can be optimally applied, depending on the situation ." In thaceiling effects [social desirability bias [First, adherence score distributions in the current Figure  and prev effects . Others  effects -8. The city bias . Simoni ity bias . Compariity bias ,12. WithSecond, the modeling of adherence score is not straightforward. As constructed its scale of measurement is discrete numerical with a possible range of 0 – 25 with skewness not amenable to a normalizing transformation. Although cut point selection for an underlying numerical measure may introduce bias in effect measurement  and may 10-HIV viral load, the adjusted odds ratio for viral suppression was 0.14  for a 6-month median adherence score greater than 5. Finally, in a third analysis of maintenance of longitudinal viral suppression, mean adherence scores were calculated for the period immediately prior to each viral load measurement, creating a score for each interval between viral load measurements. This operationalization of adherence was then fit in a GEE logit model for maintenance of viral suppression, again controlling for prior treatment experience and baseline log10-HIV viral load. The adjusted adherence odds ratio for maintaining viral suppression for a mean interval adherence score greater than 5 was 0.28  Therefore, although the adherence effect estimates were model dependent, the direction of effect was consistent and significant across models.Third, because of the observational nature of the data, measurements of adherence and HIV plasma viral load were not scheduled to occur simultaneously. Typically clinicians order viral loads every 3 – 6 months depending on clinical factors. Adherence in contrast is measured in our clinic at all routine visits. Conceptually, adherence is a construct representing a daily health behavior for which various self-report indicators have been developed and mapped to estimates of percentage adherence over a defined period or, as in the case of the Owen Clinic instrument, given interpretability primarily through demonstrated association with viral suppression. Because of the staggered nature of data accrual in the clinic, decisions must be made regarding how to line up sequential viral load and adherence measures. At a conceptual level, it is a non-trivial question to decide over how long a period an adherence measure based on a limited recall period (4 days in the case of our instrument) can be extrapolated with regard to preceding and future adherence behaviors for which the self-report data represents an imperfect indicator. In our primary analysis, we made the assumption that a given adherence assessment carried forward no longer than 90 days from the antecedent adherence measurement. Whether the observations that are not temporally matched represent truly missing observations is debatable since the very nature of the data accrual process in clinical care did not require temporal matching of adherence and viral load measurement. Because the LOCF principle has been criticized in recent years , we explDespite the limitations of self-report adherence measures, they are likely to remain the most frequent modality of adherence assessment in clinical settings. The brief self-report instrument examined in this study and in an earlier developmental study has been demonstrated to correlate with electronic drug monitoring and to be predictive of viral load responses both when administered at baseline and also when administered in longitudinal follow up of unselected patients in clinical care for HIV infection.all your pills?" (response options (numeric code): no days (0), one day (1), two days (2), three days (3), all four days (4)). Item 4 inquires "How closely did you follow your specific schedule over the last 4 days?" (response options (numeric code): never (4), some of the time (3), about half the time (2), most of the time (1), all of the time (0)). Item 5 deals with weekend adherence behavior asking "Did you skip any of the HIV medications last weekend – last Saturday or Sunday?" (response options (numeric code): no (0), yes (1)). The index score is the sum of responses to the four items with a possible range of 0 (best adherence) to 25 (poorest adherence) if each component of the regimen was dosed twice daily.A retrospective observational cohort study was conducted including all HIV-infected adults under care at the UCSD Owen Clinic between January 2003 and June 2006. Patients were included in the analyses reported here if they: 1) had at least one self report medication adherence score recorded; (2) either initiated antiretroviral therapy for the first time or began a new regimen during the study period; (3) had a plasma viral load ≥ 400 copies/ml prior to initiation of the index regimen; (4) had at least one post baseline plasma viral load; and (5) remained on the index regimen for at least 30 days. Only time on the first regimen during the study period (index regimen) is included in reported analyses. During the study period, patients on antiretroviral therapy were asked to complete, prior to meeting with their medical provider, a computer-assisted four item antiretroviral medication adherence survey [ had at lTwo outcome measures were operationally defined as: (1) time to first virologic suppression defined as HIV plasma viral load (pVL) ≤ 400 copies/ml after regimen initiation; and (2) maintenance of virologic suppression (pVL ≤ 400 copies/ml). Follow up time for each patient began with the date of initiation of the index antiretroviral regimen and ended with the earliest of the following events: (1) change or discontinuation of the index regimen; (2) last clinic visit date; or (3) end of the study period. Time to first virologic suppression on the index regimen was examined using extended Cox models incorporating time-updated adherence scores. It was confirmed that the proportional hazards assumption was met for all covariates included in the Cox models using log(t) by covariates interactions . MaintenBecause HIV plasma viral load and adherence score were not always measured on the same dates, records with missing values for adherence score after the first adherence measurement date were imputed using the last observation carried forward (LOCF) principle. Because the first adherence measurement date usually occurred after the regimen start date, records with missing early adherence scores were backfilled to the regimen start date using the score of the first adherence measurement. Adherence scores were carried forward and backfilled no more than 90 days from the temporally closest adherence measurement date. Viral load data were not carried forward.Statistical analyses were performed using Stata 10.0 . This research was approved by the University of California San Diego Human Subjects Committee (Project No. 040394)The authors declare that they have no competing interests.WCM designed the study, conducted the final analysis, and prepared the manuscript; EB and EW conducted extensive medical record review and prepared preliminary analysis of the data; CB and BC contributed to design of the study and manuscript preparation; SM advised on statistical analysis and contributed to the manuscript preparation. All authors read and approved the final manuscript.


Oxygen utilization and assembly of mtETC complexes were also determined.It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson's disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid "cytoplasmic hybrid" neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser  was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT.The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD. Sporadic Parkinson's disease (PD) is a disabling, progressive neurodegenerative disease. The neuropathological characterization of PD includes not only the loss of dopaminergic neurons in the substantia nigra and other brainstem nuclei but also the presence of cytoplasmic inclusions that form Lewy bodies and Lewy neurites in surviving neurons. ,2 DegeneNeurons prone to degeneration in PD have axons that are long and poorly myelinated and provide a massive innervation to the striatum .  MicrotubReduced or compromised axonal transport could underlie the progressive, relentless loss of dopaminergic nerve terminals in sporadic PD. Axonal transport is one aspect of mitochondrial dynamics that is critical for the distribution of functional mitochondria to distal synaptic terminals. Movement, fission, and fusion of mitochondria are functions that are also essential for neuronal survival.  Studies It is now known that genes linked to familial PD  play important roles in mitochondrial transport dynamics and function. ,20 PathoIn an effort to explore the contribution of mitochondria to features of PD pathogenesis such as reduced axonal transport, we have focused our studies on "cytoplasmic hybrid" cybrid cell lines. Cybrid cell lines were created by the fusion of donated platelets from PD or CNT volunteers with human mtDNA-free (Rho0) SH-SY5Y human neuroblastoma or NT2 teratocarcinoma cells. ,24 CybriChanges in PD cybrid cell lines correlate with changes seen in PD brain.  PD cybriNon-dividing, process-bearing, dopaminergic neuronal cells were generated from undifferentiated CNT and PD cybrids using a low concentration of staurosporine. ,33 TheseIn an effort to normalize this reduction in mitochondrial movement, we exposed PD and CNT cybrid neuronal cells to a low level near-infrared light therapy. In animal studies, low level near-infrared light therapy (LLLT) improved the outcome in retinal damage due to methanol or rotenone toxicity, -35 traumThe demographics for PD and CNT volunteers were described previously.  The HoehPD and CNT cybrid cell lines were created by fusing platelets from sporadic PD or disease-free CNT individuals with mtDNA-free Rho0 cells created in SH-SY5Y neuroblastoma cells.  Blood sa2O then treated with sterile 1 N HCl for 30 minutes, washed three times with sterile H2O and stored at room temperature (RT). Coverslip dishes were coated for 40 min.-overnight with 1.5 mg/ml poly-l-lysine  dissolved in sterile RT H2O. Dishes were rinsed twice with sterile RT H2O before adding the cell suspension.Glass bottomed 35 mm dishes  were washed with sterile HProliferating cybrid cells were harvested from flasks with 0.05% trypsin  in phosphate buffered saline  for 5 minutes at 37°C. Trypsin activity was stopped by an equal volume of CM. Cells were then centrifuged at 200 g for 5 minutes and re-suspended in CM. 40,000 cells in 2 ml CM were added to each 35 mM dish. After 18–24 hours, CM was removed and the differentiation media . A 25 μM stock solution of staurosporine  was made in dimethyl sulfoxide, frozen at -80°C. Unused aliquots were discarded after 60 days. ST (4 nM-8 nM) dilutions were made fresh in DM and replaced every 2–3 days. Differentiation was completed on day 12.® OXPHOS/PDH immunocytochemistry kit  was used to detect defective assembly of ETC complexes I-V. The manufacturer's suggested protocol was modified for coverslip dishes. Vectashield mounting medium  was added to coverslip dish after staining. Images of stained cybrid neuronal cells were created using an Olympus Fluoview laser scanning confocal microscope system . SYTO 61 (Invitrogen) at a concentration of 5 mM (30 min) was used to stain nucleic acids in the cell nucleus and cytoplasm.The MitoProfile6 cells/ml resuspended in phenol-free DMEM and separated into two sterile 35 mm culture dishes) were kept on ice. In this blinded study, one dish received LLLT (see below) and the other dish remained untreated for each cybrid line studied. After the oxygraph was calibrated according to manufacturer's instructions, 2 ml of LLLT or untreated cybrid neuronal cells were added to each chamber. The chambers were closed and cells were equilibrated (37°C). After the basal rate of respiration was established, complex V respiration was inhibited by oligomycin . Respiration was uncoupled by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone . Complex 1 was inhibited by adding rotenone at a final concentration of 1.0 μM.A two chamber oxygraph O2k  was used to measure respiration rates of dissociated CNT, PD and laser-treated CNT and PD cybrid neuronal cells according to manufacturers directions. After 12 days in DM, PD and CNT cybrid neuronal cells were harvested by rinsing with PBS. The cells  for 10 min at 37°C.  Time-lap2 (measured at the dish surface) with a continuous wave 810nm diode laser for 40 seconds . The power output of the laser was measured before each LLLT experiment with an Ohir Optronics power meter  to ensure consistent exposure. After LLLT, neuronal cells from six PD, five CNT and SH-SY5Y lines were stained with MTRed as described above. Movies  were collected at 5 to 10 minute intervals for at least 2 hours for each dish (n = 2–3 experiments +/- LLLT per cybrid line). T-tests assuming equal or unequal variance were used as needed to analyze the data.Differentiated neuronal cells from cultures of six PD and five CNT cybrid lines as well as SH-SY5Y were labeled with MtRed in phenol-free DM and 10–15 movies  were collected per dish. A second dish from each line was switched to phenol-free DM and uniformly illuminated at 50 mW/cmThe processes selected for study in all conditions were comparable in length and diameter Figure . The ranFor at least 2 hours after exposure to LLLT Table . Of the To better understand the role that mitochondrial dysfunction plays in mitochondrial movement, we double stained differentiated PD and CNT cybrid neuronal cells with antibodies to essential components of mtETC complexes I-V. The presence of staining with monoclonal antibodies directed against labile subunits of each complex  indicates that the complex is correctly assembled.  By doublAn Oxygraph 2K (Oroboros)  was usedIn this study we showed that the velocity of mitochondrial movement by axonal transport was significantly reduced in a human neuronal model of sporadic PD. Other studies have also suggested a role for reduced axonal transport in the pathogenesis of PD. Our data lends additional support for this proposition and for the role that dysfunctional mitochondria play in early PD pathogenesis. We also showed that a single treatment with near-infrared laser light can restore the velocity of mitochondrial movement in PD cybrid neuronal cells to CNT levels. LLLT with near-infrared laser light has been proven safe and effective for the treatment of complex neurological conditions like stroke in humans and rodents. ,49 Our rThe mitochondria in PD cybrids exhibit structural and functional changes that could interfere with normal movement of mitochondria by axonal transport or contribute to the differential response of PD cybrid mitochondria to LLLT. These include-1) reduced activity of complexes I and IV, 2) increased levels of oxidized and nitrated proteins, 3) insufficient ATP, 4) a swollen and abnormal morphology, 5) increased levels of modified α-synuclein, and 6) increased free/polymerized tubulin ratio. ,28,50,51A substantial body of evidence supports the proposal that complex I dysfunction plays a critical role in PD pathogenesis.  AnalysisComplex IV is also misassembled in four out of six PD cybrid lines Figure . ReducedWhile the degree of mtETC misassembly in each PD cybrid line did not correlate with mitochondrial velocity, mtETC misassembly did relate to the response of PD cybrid neuronal cells to LLLT. All the PD cybrid neuronal cells showed increased mitochondrial velocity after LLLT; however the changes in PD60 and PD61 did not consistently achieve significance Figure . CurrentThe velocity of mitochondrial movement in CNT cybrid neuronal cells was not altered by LLLT. LLLT also failed to enhance respiratory function in control animals used for a study of rotenone induced mitochondrial optic neuropathy.  Rojas etReduced ATP levels stemming from mtETC dysfunction could also limit availability of ATP for use by the motors that drive axonal transport.  Esteves The mitochondria in valinomycin-treated cerebellar neuronal cells were swollen and they blocked mitochondrial movement.  The mitoSpeculation that reduced axonal transport plays a role in PD pathogenesis has existed for some time. Our ability to assess the role of axonal transport in PD has improved recently with the development of appropriate models and methods. The outcome of our studies of human cybrid neuronal cells that contain mitochondria derived from CNT and PD volunteers illustrates that axonal transport of mitochondria is significantly reduced in PD cybrid neurites. Reduced axonal transport could underlie the loss of neurites through terminal degeneration and result in the death of dopaminergic neuronal cells in PD. The observation that a single, low level exposure to LLLT can increase the velocity of mitochondrial movement and restore it to CNT levels in PD cybrid neurites is remarkable but consistent with our current understanding of the mechanism of LLLT. In a recent publication, behavioral, metabolic and anatomic measures were improved in a dose-dependent manner in a rat model of rotenone-induced optic neuropathy.  LLLT is JS and LDT are employees and shareholders in PhotoThera, Inc. UO was an employee of PhotoThera, Inc. during 2006–2007. PAT, KMS and MKB have no competing financial interests.PAT designed experiments and conducted the axonal transport studies and wrote the manuscript; KMS prepared differentiated cybrid neuronal cells and conducted the oxygraph studies; MKB produced the cybrid lines and conducted the immunocytochemical analysis of mtETC; LDT and JS contributed to the optical and thermal design (dose selection), and reviewed the manuscript; UO contributed to the optical, thermal and experimental design and reviewed the manuscript.PD63 cybrid neuron process. Time lapse movie of MtRed-labeled mitochondria in a PD63 cybrid neuron process (pass 25) after differentiation with 6 nM staurosporine for 12 days.Click here for filePD63 cybrid neuron process 85 minutes after LLLT with 50 mW of 810 nm light for 40 seconds. Time lapse movie of MtRed-labeled mitochondria in a PD63 cybrid neuron process (pass 25) after differentiation with 6 nM staurosporine for 12 days. This movie was recorded 85 minutes after LLLT with 50 mW of 810 nm light for 40 seconds.Click here for fileCNT68 cybrid neuron process. Time lapse movie of MtRed-labeled mitochondria in a CNT68 cybrid neuron process (pass 24) after differentiation with 6 nM staurosporine for 12 days.Click here for fileCNT68 cybrid neuron process 90 minutes after LLLT with 50 mW of 810 nm light for 40 seconds. Time lapse movie of MtRed-labeled mitochondria in a CNT68 cybrid neuron process (pass 24) after differentiation with 6 nM staurosporine for 12 days. This movie was recorded 90 minutes after LLLT with 50 mW of 810 nm light for 40 seconds.Click here for file


To the Editor: Data collected from expanded surveillance study suggest that more than half of Idaho Shiga toxin–producing Escherichia coli (STEC) illnesses are caused by non-O157 serotypes. Using data from a regional medical center whose stool culture protocol included Shiga toxin testing, we predicted Idaho’s STEC incidence to be significantly higher if non-O157 STEC were routinely detected by immunoassay. Recent findings suggest that the prediction was accurate in an expanded surveillance area.Several studies have shown an increased incidence of non-O157 STEC infections in the United States. For example, a community hospital in Virginia detected non-O157 serotypes in 31% of patients with STEC from 1995–2002  for Shiga toxin . This rate was disproportionately higher than that of the remaining 6 health districts, which primarily use culture methods to screen for E. coli O157 infections in the United States suggested that annual cost associated with this pathogen is $405 million, with the cost per case varying from $26 for those who do not seek medical care to $6.2 million for a patient with fatal hemolytic uremic syndrome (HUS) (Two barriers cited for not routinely screening diarrheal stools for Shiga toxin are cost and perception of low non-O157 STEC incidence. While toxin testing is more expensive than culture testing, the potential effects of misdiagnosis may outweigh cost concerns. A study estimating the financial repercussions of E. coli O157:H7 among total STEC cases every year since 2001 (Some evidence suggests that the testing focus may be changing in the United States. We used US Census Bureau population statistics to translate reported O157:H7 and non-O157 STEC cases for each state into incidence data. Despite widespread variation in STEC testing and incidence among states, there has been a significant statistical decline in the proportion of nce 2001 ; p<0.001