Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme.

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 μm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-μs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies

Genetic and metabolic predictors of chemosensitivity in oligodendroglial neoplasms

The −1p/−19q genotype predicts chemosensitivity in oligodendroglial neoplasms, but some with intact 1p/19q also respond and not all with 1p/19q loss derive durable benefit from chemotherapy. We have evaluated the predictive and prognostic significance of pretherapy 201Tl and 18F-FDG SPECT and genotype in 38 primary and 10 recurrent oligodendroglial neoplasms following PCV chemotherapy. 1p/19q loss was seen in 8/15 OII, 6/15 OAII, 7/7 OIII, 3/11 OAIII and was associated with response (Fisher-Exact: P=0.000) and prolonged progression-free (log-rank: P=0.002) and overall survival (OS) (log-rank: P=0.0048). Response was unrelated to metabolism, with tumours with high or low metabolism showing response. Increased 18F-FDG or 201Tl uptake predicted shorter progression-free survival (PFS) in the series (log-rank: 201Tl P=0.0097, 18F-FDG P=0.0170) and in cases with or without the −1p/−19q genotype. Elevated metabolism was associated with shorter OS in cases with intact 1p/19q (log-rank: 18F-FDG P=0.0077; 201Tl P=0.0004) and shorter PFS in responders (log-rank: 18F-FDG P=0.005; 201Tl P=0.0132). 201Tl uptake and 1p/19q loss were independent predictors of survival in multivariate analysis. In this initial study, 201Tl and 18F-FDG uptake did not predict response to PCV, but may be associated with poor survival following therapy irrespective of genotype. This may be clinically useful warranting further study

MGMT promoter hypermethylation is a frequent, early, and consistent event in astrocytoma progression, and not correlated with TP53 mutation

Hypermethylation of the MGMT gene promoter and mutation of the TP53 tumor-suppressor gene are frequently present in diffuse astrocytomas. However, there is only anecdotal information about MGMT methylation status and TP53 mutations during progression of low-grade diffuse astrocytoma (AII) to anaplastic astrocytoma (AIII) and secondary glioblastoma (sGB). In this study biopsy specimens from 51 patients with astrocytic tumors with radiologically proved progression from low to high-grade malignancy were investigated for the presence and consistency of MGMT promoter hypermethylation and TP53 mutations. For 27 patients biopsy samples both of primary tumors and their recurrences were available. For the other 24 patients histology of either the low-grade lesion or the high-grade recurrence was available. It was found that MGMT promoter hypermethylation and TP53 mutations are both frequent and early events in the progression of astrocytomas and that their status is consistent over time. No correlation was found between MGMT methylation status and the presence of TP53 mutations. In addition, no correlation was found between MGMT promoter hypermethylation and the type of TP53 mutations. These results argue against the putative TP53 G:C>A:T transition mutations suggested to occur preferentially in MGMT hypermethylated tumors

Genomic aberrations associated with outcome in anaplastic oligodendroglial tumors treated within the EORTC phase III trial 26951

Despite similar morphological aspects, anaplastic oligodendroglial tumors (AOTs) form a heterogeneous clinical subgroup of gliomas. The chromosome arms 1p/19q codeletion has been shown to be a relevant biomarker in AOTs and to be perfectly exclusive from EGFR amplification in gliomas. To identify new genomic regions associated with prognosis, 60 AOTs from the EORTC trial 26951 were analyzed retrospectively using BAC-array-based comparative genomic hybridization. The data were processed using a binary tree method. Thirty-three BACs with prognostic value were identified distinguishing four genomic subgroups of AOTs with different prognosis (p < 0.0001). Type I tumors (25%) were characterized by: (1) an EGFR amplification, (2) a poor prognosis, (3) a higher rate of necrosis, and (4) an older age of patients. Type II tumors (21.7%) had: (1) loss of prognostic BACs located on 1p tightly associated with 19q deletion, (2) a longer survival, (3) an oligodendroglioma phenotype, and (4) a frontal location in brain. Type III AOTs (11.7%) exhibited: (1) a deletion of prognostic BACs located on 21q, and (2) a short survival. Finally, type IV tumors (41.7%) had different genomic patterns and prognosis than type I, II and III AOTs. Multivariate analysis showed that genomic type provides additional prognostic data to clinical, imaging and pathological features. Similar results were obtained in the cohort of 45 centrally reviewed–validated cases of AOTs. Whole genome analysis appears useful to screen the numerous genomic abnormalities observed in AOTs and to propose new biomarkers particularly in the non-1p/19q codeleted AOTs

Identification of two contiguous minimally deleted regions on chromosome 1p36.31–p36.32 in oligodendroglial tumours

Loss of the short arm of chromosome 1 is a hallmark of oligodendroglial tumours (OTs). Deletion mapping studies in OTs have revealed multiple commonly deleted regions on chromosome 1p, suggesting that there are more than one tumour suppressor gene. To map critical deletion regions on 1p, a series of 25 OTs were examined for loss of heterozygosity (LOH) on 19 polymorphic markers across the 1p arm using microsatellite analysis. Our study revealed that 60% of tumours had LOH of all informative markers on 1p and identified one tumour showing LOH at telomeric markers only. Since this deletion region lies in one of the critical deletion intervals defined previously, we then screened another series of 27 OTs specifically at 1p36.3 for LOH using nine polymorphic markers. A total of 12% (six out of 52) of tumours were found to carry interstitial deletions. The allelic status and the deletion breakpoints in these tumours with interstitial deletion were further verified by fluorescent in situ hybridisation. The small overlapping intervals facilitated the delineation of two contiguous minimally deleted regions of 0.76 Mb, defined by D1S468 and D1S2845, and of 0.41 Mb, bound by D1S2893 and D1S1608, on 1p36.31–36.32. Based on current reference human genome sequence these deletion regions have been sequenced almost to entirety and contain eight annotated genes. TP73, DFFB and SHREW1 are the only known genes located in these deletion regions, while the others are uncharacterised novel genes. In conclusion, our study has narrowed down the critical tumour suppressor loci on 1p36.3, in which two minimally deleted regions are mapped, and markedly reduced the number of candidate genes to be screened for their involvement in OT development

EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase autoinhibitory mechanism

Tumor cells often subvert normal regulatory mechanisms of signal transduction. This study shows this principle by studying yet uncharacterized mutants of the epidermal growth factor receptor (EGFR) previously identified in glioblastoma multiforme, which is the most aggressive brain tumor in adults. Unlike the well-characterized EGFRvIII mutant form, which lacks a portion of the ligand-binding cleft within the extracellular domain, EGFRvIVa and EGFRvIVb lack internal segments distal to the intracellular tyrosine kinase domain. By constructing the mutants and by ectopic expression in naive cells, we show that both mutants confer an oncogenic potential in vitro, as well as tumorigenic growth in animals. The underlying mechanisms entail constitutive receptor dimerization and basal activation of the kinase domain, likely through a mechanism that relieves a restraining molecular fold, along with stabilization due to association with HSP90. Phosphoproteomic analyses delineated the signaling pathways preferentially engaged by EGFRvIVb-identified unique substrates. This information, along with remarkable sensitivities to tyrosine kinase blockers and to a chaperone inhibitor, proposes strategies for pharmacological interception in brain tumors harboring EGFRvIV mutations.Goldhirsh FoundationNational Cancer Institute (U.S.) (CA118705)National Cancer Institute (U.S.) (CA141556)National Cancer Institute (U.S.) (U54-CA112967

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell line

The Accuracy of Survival Time Prediction for Patients with Glioma Is Improved by Measuring Mitotic Spindle Checkpoint Gene Expression

Identification of gene expression changes that improve prediction of survival time across all glioma grades would be clinically useful. Four Affymetrix GeneChip datasets from the literature, containing data from 771 glioma samples representing all WHO grades and eight normal brain samples, were used in an ANOVA model to screen for transcript changes that correlated with grade. Observations were confirmed and extended using qPCR assays on RNA derived from 38 additional glioma samples and eight normal samples for which survival data were available. RNA levels of eight major mitotic spindle assembly checkpoint (SAC) genes (BUB1, BUB1B, BUB3, CENPE, MAD1L1, MAD2L1, CDC20, TTK) significantly correlated with glioma grade and six also significantly correlated with survival time. In particular, the level of BUB1B expression was highly correlated with survival time (p<0.0001), and significantly outperformed all other measured parameters, including two standards; WHO grade and MIB-1 (Ki-67) labeling index. Measurement of the expression levels of a small set of SAC genes may complement histological grade and other clinical parameters for predicting survival time

A gene encoding an abscisic acid biosynthetic enzyme (LsNCED4) collocates with the high temperature germination locus Htg6.1 in lettuce (Lactuca sp.)

Thermoinhibition, or failure of seeds to germinate when imbibed at warm temperatures, can be a significant problem in lettuce (Lactuca sativa L.) production. The reliability of stand establishment would be improved by increasing the ability of lettuce seeds to germinate at high temperatures. Genes encoding germination- or dormancy-related proteins were mapped in a recombinant inbred line population derived from a cross between L. sativa cv. Salinas and L. serriola accession UC96US23. This revealed several candidate genes that are located in the genomic regions containing quantitative trait loci (QTLs) associated with temperature and light requirements for germination. In particular, LsNCED4, a temperature-regulated gene in the biosynthetic pathway for abscisic acid (ABA), a germination inhibitor, mapped to the center of a previously detected QTL for high temperature germination (Htg6.1) from UC96US23. Three sets of sister BC3S2 near-isogenic lines (NILs) that were homozygous for the UC96US23 allele of LsNCED4 at Htg6.1 were developed by backcrossing to cv. Salinas and marker-assisted selection followed by selfing. The maximum temperature for germination of NIL seed lots with the UC96US23 allele at LsNCED4 was increased by 2–3°C when compared with sister NIL seed lots lacking the introgression. In addition, the expression of LsNCED4 was two- to threefold lower in the former NIL lines as compared to expression in the latter. Together, these data strongly implicate LsNCED4 as the candidate gene responsible for the Htg6.1 phenotype and indicate that decreased ABA biosynthesis at high imbibition temperatures is a major factor responsible for the increased germination thermotolerance of UC96US23 seeds