Candida spp. cause both local and disseminated infections in immunocompromised patients. Bloodstream infections of Candida spp., known as "candidemia," are associated with a high mortality rate (40%), which is mainly attributed to the long diagnostic time required by blood culture. We introduce a diagnostic platform based on T2 magnetic resonance (T2MR), which is capable of sensitive and rapid detection of fungal targets in whole blood. In our approach, blood-compatible polymerase chain reaction is followed by hybridization of the amplified pathogen DNA to capture probe-decorated nanoparticles. Hybridization yields nanoparticle microclusters that cause large changes in the sample's T2MR signal. With this T2MR-based method, Candida spp. can be detected directly in whole blood, thus eliminating the need for analyte purification. Using a small, portable T2MR detection device, we were able to rapidly, accurately, and reproducibly detect five Candida species within human whole blood with a limit of detection of 1 colony-forming unit/ml and a time to result of <3 hours. Spiked blood samples showed 98% positive agreement and 100% negative agreement between T2MR and blood culture. Additionally, performance of the assay was evaluated on 21 blinded clinical specimens collected serially. This study shows that the nanoparticle- and T2MR-based detection method is rapid and amenable to automation and offers clinicians the opportunity to detect and identify multiple human pathogens within hours of sample collection.

Cell-free RNA (cfRNA) is a promising analyte for cancer detection. However, a comprehensive assessment of cfRNA in individuals with and without cancer has not been conducted. We perform the first transcriptome-wide characterization of cfRNA in cancer (stage III breast [n = 46], lung [n = 30]) and non-cancer (n = 89) participants from the Circulating Cell-free Genome Atlas (NCT02889978). Of 57,820 annotated genes, 39,564 (68%) are not detected in cfRNA from non-cancer individuals. Within these low-noise regions, we identify tissue- and cancer-specific genes, defined as “dark channel biomarker” (DCB) genes, that are recurrently detected in individuals with cancer. DCB levels in plasma correlate with tumor shedding rate and RNA expression in matched tissue, suggesting that DCBs with high expression in tumor tissue could enhance cancer detection in patients with low levels of circulating tumor DNA. Overall, cfRNA provides a unique opportunity to detect cancer, predict the tumor tissue of origin, and determine the cancer subtype.

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

We report construction of a portable nuclear magnetic resonance sensor with a single-sided open probe design. The resulting magnetic field inhomogeneity is compensated by a pulse sequence that takes advantage of parallel inhomogeneity in the applied radio frequency field. We can thereby acquire fluorine-19 spectra of liquid fluorocarbons with 8 parts per million resolution, surmounting the long-standing obstacle of obtaining chemical shift information with open probe instruments.

SummarySustained caloric restriction (CR) extends lifespan in animal models but the mechanism and primary tissue target(s) have not been identified. Gene expression changes with aging and CR were examined in both heart and white adipose tissue (WAT) of Fischer 344 (F344) male rats using Affymetrix® RAE 230 arrays and validated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on 18 genes. As expected, age had a substantial effect on transcription on both tissues, although only 21% of cardiac age-associated genes were also altered in WAT. Gene set enrichment analysis revealed coordinated small magnitude changes in ribosomal, proteasomal, and mitochondrial genes with similarities in aging between heart and WAT. CR had very different effects on these two tissues at the transcriptional level. In heart, very few age-associated expression changes were affected by CR, while in WAT, CR suppressed a substantial subset of the age-associated changes. Genes unaltered by aging but altered by CR were identified in WAT but not heart. Most interestingly, we identified a gene expression signature associated with mammalian target of rapamycin (mTOR) activity that was down-regulated with age but preserved by CR in both WAT and heart. In addition, lipid metabolism genes, particularly those associated with peroxisome proliferator-activated receptor γ γ γ γ (PPARγ γ γ γ )-mediated adipogenesis were reduced with age but preserved with CR in WAT. These results highlight tissue-specific differences in the gene expression response to CR and support a role for CR-mediated preservation of mTOR activity and adipogenesis in aging WAT.

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The functional diversity of voltage-gated K+ channels may be partially determined by the mechanisms that permit or limit the assembly of molecularly diverse K+ channel subunits. To determine possible amino acid sequence domains required for subunit assembly and expression, we have constructed 15 N- and C-terminal interstitial or truncation deletion mutations in mKv1.1 (MBK1), a mouse Shaker-like K+ channel. We injected Xenopus oocytes with cRNA encoding each of these mutants and coinjected each mutant cRNA with cRNA for wild-type mKv1.3, another mouse Shaker-like K+ channel that can form heteromultimers with mKv1.1. We found that the last five amino acids of the C-terminus of mKv1.1 contribute to functional expression by (1) rescuing the function of mutants with a large truncation of the C-terminus (delta 424–495), and (2) contributing to the slow inactivation kinetics (time constant of 2– 3 sec) of wild-type mKv1.1 whole-cell K+ currents. All C-terminal deletion mutants were able to express at least as heteromultimers with mKv1.3, suggesting that the C-terminus is not required for channel assembly. In contrast, nine different interstitial or truncation mutants in which part of a highly conserved, large (80–99 amino acid residues) domain within the N-terminus had been deleted were unable to express either homomultimers or heteromultimers. The relatively small sizes and nonoverlapping distributions of the interstitial deletions enable us to suggest that the structural integrity of this entire N- terminal domain is required for subunit assembly and functional expression of this and probably other Shaker-like K+ channel proteins.

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