Sequence alignment of mass peaks matching to ATP5B.

<p>Clustal alignment of the ATP5B sequence with trypsin digestion peptides identified by MS with a matching sequence of confidence 85% or greater.</p

Vimentin ELISA.

<p>Levels of antibodies to vimentin were determined at least in duplicate and individual patient (open diamonds) and control (closed triangles) values are plotted on the graph. The defined cut-off value of two standard deviations above the mean of the control group (excluding the outlier) is depicted by the hashed horizontal line. The index MM patient sample (Patient 145) result is indicated by the closed black diamond symbol. There was no significant difference (ns) between the groups.</p

Immunohistochemical tissue localisation of β-F1-ATPpase.

<p>Immunohistochemical staining of representative MM samples using anti-β-F1-ATPpase antibody diluted 1∶100 (A) Positive stained MM tumour sample, arrows indicate representative groups of tumour cells; over 90% of the tumour cells have been stained. (B) Non stained MM tumour sample with positively stained macrophage (indicated by arrow); less than 10% of tumour cells have been stained. (C) Positively stained MM cells present in a pleural effusion sample. Arrows indicate representative positive stained MM cells; over 90% of the tumour cells have been stained. (D) Non stained MM tumour cells in a MM patient pleural effusion; less than 10% of tumour cells have been stained. Photomicrographs were taken at a ×200 magnification.</p

Immunohistochemicallocalisation of vimentin.

<p>Immunohistochemical staining of representative MM samples using anti-vimentin antibody diluted 1∶500 (A) Positive stained MM tumour sample, arrows indicate representative groups of tumour cells; over 90% of the tumour cells have been stained (B) Non stained MM tumour sample with positively stained stroma. Arrows indicate representative groups of non-staining tumour cells; less than 10% of tumour cells have been stained. (C) Positively stained MM cells present in a pleural effusion sample. Arrows indicate representative positive stained MM cells; over 90% of the tumour cells have been stained. (D) Non stained MM tumour cells in a MM patient pleural effusion. Arrows indicate representative non-staining tumour cells; less than 10% of tumour cells have been stained. Photomicrographs were taken at ×200 magnification.</p

ß-F1-ATPase ELISA.

<p>Levels of antibodies to ß-F1-ATPase were determined at least in duplicate and individual patient (open diamonds) and control (closed triangles) values are plotted on the graph. The defined cut-off value of two standard deviations above the mean of the control group is depicted by the hashed horizontal line. The index MM patient sample (Patient 145) result is indicated by the closed black diamond symbol. The MM patients indicated by grey symbols were previously identified as being immunopositive to ß-F1-ATPase by SEREX. There was no significant difference (ns) between the groups.</p

Survival of MM patients dichotomized on antibody reactivity.

<p>Kaplan–Meier survival curves of overall survival (months) from diagnosis for patients with (A) anti-vimentin and (B) anti-β-F1-ATPase antibody levels dichotomized below (thick line) and above the median (thin line).</p

Western Immunoblots of MM patient serum with MM tumour cell line lysates.

<p>(A) Representative serum samples from individual MM patients. Patients segregated into short, medium and long term survivors. Patient identification number indicated above each lane. (B) Western immunoblot of cell lysates from five different MM cell lines and the A549 lung adenocarcinoma, U2OS osteosarcoma and HT29 colon carcinoma cell lines incubated with serum from a MM Patient 145. Serum was diluted 1∶100. Molecular weights (MW) depicted to the left of figures.</p

Two Dimensional Polyacrylamide Gel Electrophoresis.

<p>The Tris-soluble fraction of the JU77 cell lysate was separated by 2D electrophoresis and proteins stained with Coomassie Blue. Proteins from a parallel gel were transferred to PDVF and incubated with serum from MM Patient 145 diluted 1∶100. Two densely stained IgG reactive spots were defined by the approximate pI range 5 to 7 and molecular weight range 50 to 60 kDa, indicated by the circle. Molecular weights (MW) depicted to the left of figures; isoelectric point (pI) to the top of figures.</p

Clinical characteristics and antibody levels for study subjects.

1<p>– Short term survival group; survival <12 months.</p>2<p>– Long term survival group; survival >12 months.</p>3<p>- median ± 95% confidence intervals.</p

Mitochondrial Dysfunction in <em>Pten</em> Haplo-Insufficient Mice with Social Deficits and Repetitive Behavior: Interplay between Pten and p53

<div><p>Etiology of aberrant social behavior consistently points to a strong polygenetic component involved in fundamental developmental pathways, with the potential of being enhanced by defects in bioenergetics. To this end, the occurrence of social deficits and mitochondrial outcomes were evaluated in conditional <em>Pten</em> (Phosphatase and tensin homolog) haplo-insufficient mice, in which only one allele was selectively knocked-out in neural tissues. <em>Pten</em> mutations have been linked to Alzheimer's disease and syndromic autism spectrum disorders, among others. By 4–6 weeks of age, Pten insufficiency resulted in the increase of several mitochondrial Complex activities (II–III, IV and V) not accompanied by increases in mitochondrial mass, consistent with an activation of the PI3K/Akt pathway, of which Pten is a negative modulator. At 8–13 weeks of age, <em>Pten</em> haplo-insufficient mice did not show significant behavioral abnormalities or changes in mitochondrial outcomes, but by 20–29 weeks, they displayed aberrant social behavior (social avoidance, failure to recognize familiar mouse, and repetitive self-grooming), macrocephaly, increased oxidative stress, decreased cytochrome <em>c</em> oxidase (CCO) activity (50%) and increased mtDNA deletions in cerebellum and hippocampus. Mitochondrial dysfunction was the result of a downregulation of p53-signaling pathway evaluated by lower protein expression of p21 (65% of controls) and the CCO chaperone SCO2 (47% of controls), two p53-downstream targets. This mechanism was confirmed in Pten-deficient striatal neurons and, HCT 116 cells with different p53 gene dosage. These results suggest a unique pathogenic mechanism of the Pten-p53 axis in mice with aberrant social behavior: loss of Pten (via p53) impairs mitochondrial function elicited by an early defective assembly of CCO and later enhanced by the accumulation of mtDNA deletions. Consistent with our results, (i) SCO2 deficiency and/or CCO activity defects have been reported in patients with learning disabilities including autism and (ii) mutated proteins in ASD have been found associated with p53-signaling pathways.</p> </div