Prospective cohort study of radiotherapy with concomitant and adjuvant temozolomide chemotherapy for glioblastoma patients with no or minimal residual enhancing tumor load after surgery

Survival of glioblastoma patients has been linked to the completeness of surgical resection. Available data, however, were generated with adjuvant radiotherapy. Data confirming that extensive cytoreduction remains beneficial to patients treated with the current standard, concomitant temozolomide radiochemotherapy, are limited. We therefore analyzed the efficacy of radiochemotherapy for patients with little or no residual tumor after surgery. In this prospective, non-interventional multicenter cohort study, entry criteria were histological diagnosis of glioblastoma, small enhancing or no residual tumor on post-operative MRI, and intended temozolomide radiochemotherapy. The primary study objective was progression-free survival; secondary study objectives were survival and toxicity. Furthermore, the prognostic value of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation was investigated in a subgroup of patients. One-hundred and eighty patients were enrolled. Fourteen were excluded by patient request or failure to initiate radiochemotherapy. Twenty-three patients had non-evaluable post-operative imaging. Thus, 143 patients qualified for analysis, with 107 patients having residual tumor diameters ≤1.5 cm. Median follow-up was 24.0 months. Median survival or patients without residual enhancing tumor exceeded the follow-up period. Median survival was 16.9 months for 32 patients with residual tumor diameters >0 to ≤1.5 cm (95% CI: 13.3–20.5, p = 0.039), and 13.9 months (10.3–17.5, overall p < 0.001) for 36 patients with residual tumor diameters >1.5 cm. Patient age at diagnosis and extent of resection were independently associated with survival. Patients with MGMT promoter methylated tumors and complete resection made the best prognosis. Completeness of resection acts synergistically with concomitant and adjuvant radiochemotherapy, especially in patients with MGMT promoter methylation

Evidence for Loss of a Partial Flagellar Glycolytic Pathway during Trypanosomatid Evolution

Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed

Moonlighting enzymes in parasitic protozoa

Enzymes moonlight in a non-enzymatic capacity in a diverse variety of cellular processes. The discovery of these non-enzymatic functions is generally unexpected, and moonlighting enzymes are known in both prokaryotes and eukaryotes. Importantly, this unexpected multi-functionality indicates that caution might be needed on some occasions in interpreting phenotypes that result from the deletion or gene-silencing of some enzymes, including some of the best known enzymes from classic intermediary metabolism. Here, we provide an overview of enzyme moonlighting in parasitic protists. Unequivocal and putative examples of moonlighting are discussed, together with the possibility that the unusual biological characteristics of some parasites either limit opportunities for moonlighting to arise or perhaps contribute to the evolution of novel proteins with clear metabolic ancestry

Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression

It is widely believed that long-term depression (LTD) and its counterpart, long-term potentiation (LTP), involve mechanisms that are crucial for learning and memory. However, LTD is difficult to induce in adult cortex for reasons that are not known. Here we show that LTD can be readily induced in adult cortex by the activation of NMDA receptors (NMDARs), after inhibition of glutamate uptake. Interestingly there is no need to activate synaptic NMDARs to induce this LTD, suggesting that LTD is triggered primarily by extrasynaptic NMDA receptors. We also find that de novo LTD requires the activation of NR2B-containing NMDAR, whereas LTP requires activation of NR2A-containing NMDARs. Surprisingly another form of LTD, depotentiation, requires activation of NR2A-containing NMDARs. Therefore, NMDARs with different synaptic locations and subunit compositions are involved in various forms of synaptic plasticity in adult cortex

Generation of Tb<i>GAPDHL</i> and Tb<i>PGKL</i> procyclic null mutants.

<p>(<b>A</b>) Generation of Tb<i>GAPDHL</i> null mutants. (<b>B</b>) Generation of Tb<i>PGKL</i> null mutants. Cartoon schematics denote gene loci annotated with HindIII (H) restriction sites for (i) wild-type loci; (ii) loci following gene disruption and (iii) following endogenous gene-tagging with GFP (Tb<i>GAPDHL</i> only). Southern analysis of genomic DNA digested overnight at 37°C with HindIII shows blots probed sequentially with either coding sequence from the targeted gene (probe o) or sequence from the 3′ intergenic region (probe u). Relative positions of the probes are shown in the cartoon schematics. In (<b>A</b>) the order of lanes is 1, wild-type <i>GAPDHL</i>^+/+<i>T. brucei</i>; 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4-5, <i>GAPDHL</i>^+/− heterozygotes from lanes 2 and 3, respectively, in which endogenous tagging of the remaining wild-type allele results in expression of a recombinant e<i>GFP:GAPDHL</i>; 6, a <i>GAPDHL^−/−</i> mutant obtained from the stable transformation of the phleomycin-resistant heterozygote cells from lane 2. In (<b>B</b>) the order of lanes is 1, wild-type <i>PGKL</i>^+/+, 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4–5, independently obtained <i>PGKL</i>^−/− mutants derived from the stable transformation of heterozygous cell lines analyzed in lanes 2 and 3, respectively.</p

Flagellar localization of <i>Tb</i>PGKL.

<p>(<b>A</b>) Indirect immunofluorescence using monoclonal antibody BB2 reveals axonemal localization of Ty::<i>Tb</i>PGKL in detergent-extracted procyclic <i>T. brucei</i> cytoskeletons. Cytoskeletons were stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect mitochondrial (kinetoplast, K) and nuclear (N) DNA. The inset shows how the indirect immunofluorescence signal extends close to the kinetoplast, consistent with axoneme association. Scale bar denotes 5 µm. (<b>B</b>) Immunoblot analysis of detergent- and NaCl-extracted flagella isolated from procyclic cells expressing Ty::<i>Tb</i>PGK-like protein using BB2 detects a single band of the expected molecular mass.</p

PFR localization of <i>Tb</i>GAPDHL.

<p>(<b>A</b>) Localization of GFP::<i>Tb</i>GAPDHL in procyclic <i>T. brucei</i> cells. (<b>B</b>) Indirect immunofluorescence of detergent-extracted cytoskeletons using the monoclonal antibody L8C4 to detect the major PFR protein PFR2 suggests GFP::<i>Tb</i>GAPDHL is a novel PFR component. Insets 1 and 2 indicate that at the proximal end of the flagellum PFR2 incorporation into flagellar skeleton does not begin prior to GFP::<i>Tb</i>GAPDHL incorporation – <i>cf</i> inset 2 in (<b>C</b>). (<b>C</b>) A lack of co-localization in indirect immunofluorescence of cytoskeletons using the monoclonal antibody L3B2 indicates GFP::<i>Tb</i>GAPDHL is not a cytoplasmic FAZ component: inset 1 indicates flagellar GFP::<i>Tb</i>GAPDHL fluorescence extends beyond the end of the cell body as denoted by L3B2 labelling of the cytoplasmic FAZ filament; inset 2 highlights how assembly the cytoplasmic FAZ filament detected by L3B2 initiates before assembly of GFP::<i>Tb</i>GAPDHL into the flagellar architecture. (<b>D</b>) Change in YFP::<i>Tb</i>GAPDHL localization in Tb<i>CaM</i> RNAi mutants: following RNAi induction and failure of PFR assembly YFP::<i>Tb</i>GAPDHL co-localizes with aggregates containing PFR2 protein (detected by indirect immunofluorescence with monoclonal antibody L8C4). (<b>E</b>) PFR localization of GFP::<i>Tb</i>GAPDHL is retained in <i>snl-2</i> RNAi mutants; detergent extracted cytoskeletons were also stained for indirect immunofluorescence with L13D6 to highlight failure to incorporate either PFR1 or PFR2, the two major PFR components, into the flagellar architecture. DIC, differential interference contrast; N, nucleus; K, kinetoplast. Scale bars denote 5 µm.</p

Sequence alignment of trypanosomatid GAPDHL proteins with authentic GAPDH.

<p>The alignment was built using MUSCLE and sequences are named with species abbreviations (Tb =  <i>T. brucei</i>, Tc =  <i>T. cruzi</i>, Lm =  <i>L. major</i>, Bs =  <i>B. saltans</i>, Gt =  <i>Geobacillus stearothermophilus</i>, Mb =  <i>Mycobacterium bovis</i>, Hs =  <i>Homo sapiens</i>, Ce =  <i>Caenorhabditis elegans</i>, Mj =  <i>Methanocaldococcus jannaschii</i>, Pt =  <i>Picrophilus torridus</i>) followed by a locus code (kinetoplastid sequences) or UniProt accession. For kinetoplastid sequences, cGAPDH indicates the cytosolic isoform and gGAPDH the glycosomal enzyme. Only one each of the tandem copies of gGAPDH is shown for each trypanosomatid. Residues mentioned in the text are highlighted as white on purple.</p

Range and mean percentage amino acid identities between GAPDH(-like) groups in trypanosomatids.

<p>Each group contains sequences (eliminating tandem duplicates) from <i>T. brucei, T. cruzi, T. vivax, L. braziliensis, L. mexicana, L. major, L. infantum, L. tarentolae</i>, and <i>Endotrypanum monterogeii</i>.</p