Enhancing metabolite coverage in MALDI-MSI using laser post-ionisation (MALDI-2).

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) of metabolites can reveal how metabolism is altered throughout heterogeneous tissues. Here negative ion mode MALDI-MSI has been coupled with laser post-ionisation (MALDI-2) and applied to the MSI of low molecular weight (LMW) metabolites (

Experimental and Data Analysis Considerations for Three-Dimensional Mass Spectrometry Imaging in Biomedical Research.

Mass spectrometry imaging (MSI) enables the visualization of molecular distributions on complex surfaces. It has been extensively used in the field of biomedical research to investigate healthy and diseased tissues. Most of the MSI studies are conducted in a 2D fashion where only a single slice of the full sample volume is investigated. However, biological processes occur within a tissue volume and would ideally be investigated as a whole to gain a more comprehensive understanding of the spatial and molecular complexity of biological samples such as tissues and cells. Mass spectrometry imaging has therefore been expanded to the 3D realm whereby molecular distributions within a 3D sample can be visualized. The benefit of investigating volumetric data has led to a quick rise in the application of single-sample 3D-MSI investigations. Several experimental and data analysis aspects need to be considered to perform successful 3D-MSI studies. In this review, we discuss these aspects as well as ongoing developments that enable 3D-MSI to be routinely applied to multi-sample studies.

Rapid Identification of Ischemic Injury in Renal Tissue by Mass-Spectrometry Imaging.

The increasing analytical speed of mass-spectrometry imaging (MSI) has led to growing interest in the medical field. Acute kidney injury is a severe disease with high morbidity and mortality. No reliable cut-offs are known to estimate the severity of acute kidney injury. Thus, there is a need for new tools to rapidly and accurately assess acute ischemia, which is of clinical importance in intensive care and in kidney transplantation. We investigated the value of MSI to assess acute ischemic kidney tissue in a porcine model. A perfusion model was developed where paired kidneys received warm (severe) or cold (minor) ischemia ( n = 8 per group). First, ischemic tissue damage was systematically assessed by two blinded pathologists. Second, MALDI-MSI of kidney tissues was performed to study the spatial distributions and compositions of lipids in the tissues. Histopathological examination revealed no significant difference between kidneys, whereas MALDI-MSI was capable of a detailed discrimination of severe and mild ischemia by differential expression of characteristic lipid-degradation products throughout the tissue within 2 h. In particular, lysolipids, including lysocardiolipins, lysophosphatidylcholines, and lysophosphatidylinositol, were dramatically elevated after severe ischemia. This study demonstrates the significant potential of MSI to differentiate and identify molecular patterns of early ischemic injury in a clinically acceptable time frame. The observed changes highlight the underlying biochemical processes of acute ischemic kidney injury and provide a molecular classification tool that can be deployed in assessment of acute ischemic kidney injury.

Prospective, randomized, double-blind assessment of topical bakuchiol and retinol for facial photoageing.

BACKGROUND: Bakuchiol is a phytochemical that has demonstrated cutaneous antiageing effects when applied topically. Early studies have suggested that bakuchiol is a functional analogue of topical retinoids, as both compounds have been shown to induce similar gene expression in the skin and lead to improvement of cutaneous photodamage. No in vivo studies have compared the two compounds for efficacy and side-effects. OBJECTIVES: To compare the clinical efficacy and side-effect profiles of bakuchiol and retinol in improving common signs of cutaneous facial ageing. METHODS: This was a randomized, double-blind, 12-week study in which 44 patients were asked to apply either bakuchiol 0·5% cream twice daily or retinol 0·5% cream daily. A facial photograph and analytical system was used to obtain and analyse high-resolution photographs of patients at 0, 4, 8 and 12 weeks. Patients also completed tolerability assessment questions to review side-effects. During study visits, a board-certified dermatologist, blinded to study group assignments, graded pigmentation and redness. RESULTS: Bakuchiol and retinol both significantly decreased wrinkle surface area and hyperpigmentation, with no statistical difference between the compounds. The retinol users reported more facial skin scaling and stinging. CONCLUSIONS: Our study demonstrates that bakuchiol is comparable with retinol in its ability to improve photoageing and is better tolerated than retinol. Bakuchiol is promising as a more tolerable alternative to retinol.

Strategies for managing multi-patient 3D mass spectrometry imaging data.

Mass spectrometry imaging (MSI) has emerged as a powerful tool in biomedical research to reveal the localization of a broad scale of compounds ranging from metabolites to proteins in diseased tissues, such as malignant tumors. MSI is most commonly used for the two-dimensional imaging of tissues from multiple patients or for the three-dimensional (3D) imaging of tissue from a single patient. These applications are potentially introducing a sampling bias on a sample or patient level, respectively. The aim of this study is therefore to investigate the consequences of sampling bias on sample representativeness and on the precision of biomarker discovery for histological grading of human bladder cancers by MSI. We therefore submitted formalin-fixed paraffin-embedded tissues from 14 bladder cancer patients with varying histological grades to 3D analysis by matrix-assisted laser desorption/ionization (MALDI) MSI. We found that, after removing 20% of the data based on novel outlier detection routines for 3D-MSI data based on the evaluation of digestion efficacy and z-directed regression, on average 33% of a sample has to be measured in order to obtain sufficient coverage of the existing biological variance within a tissue sample. SIGNIFICANCE: In this study, 3D MALDI-MSI is applied for the first time on a cohort of bladder cancer patients using formalin-fixed paraffin-embedded (FFPE) tissue of bladder cancer resections. This work portrays the reproducibility that can be achieved when employing an optimized sample preparation and subsequent data evaluation approach. Our data shows the influence of sampling bias on the variability of the results, especially for a small patient cohort. Furthermore, the presented data analysis workflow can be used by others as a 3D FFPE data-analysis pipeline working on multi-patient 3D-MSI studies.

Laser post-ionisation combined with a high resolving power orbitrap mass spectrometer for enhanced MALDI-MS imaging of lipids.

Coupling laser post-ionisation with a high resolving power MALDI Orbitrap mass spectrometer has realised an up to ∼100-fold increase in the sensitivity and enhanced the chemical coverage for MALDI-MS imaging of lipids relative to conventional MALDI. This could constitute a major breakthrough for biomedical research.

Organocatalytic enantioselective desymmetrisation.

Organocatalytic enantioselective desymmetrisation of achiral or meso compounds is a powerful strategy for the construction of enantiomerically enriched complex molecules, often with multiple stereocentres and in high selectivities. Recent years have seen increasing use of organocatalysts in desymmetrisation methodology, in contrast to traditional metal- or enzyme-catalysed reactions, with many impressive advances made in the current decade. This review will provide an overview of the field since 2010, with the aim of highlighting both the practical applications and elegance of enantioselective desymmetrisation to the wider synthetic community.

A multidisciplinary combinatorial approach for tuning promising hydrogen storage materials towards automotive applications.

HyStorM is a multidisciplinary hydrogen-storage project aiming to synthesise and tune materials hydrogen storage properties for automotive applications. Firstly, unique high-throughput combinatorial thin-film technologies are used to screen materials' hydrogen storage properties. Then promising thin-film candidate compositions are synthesised and examined in the bulk. In this paper, we report on our results within the ternary compositions Mg-Ti-B and Ca-Ti-B. Primary screening of the Mg-Ti-B ternary identified a high capacity hotspot corresponding to Mg0.36Ti0.06B0.58, with 10.6 wt% H2 capacity. Partial reversibility has been observed for this material in the thin-film. Bulk Ti-doped Mg(BH4)2 composites show rehydrogenation to MgH2 under the conditions used. The synthesised thin-film Ca-Ti-B ternary showed only low hydrogen storage capacities. In the bulk, Ti-doping experiments on Ca(BH4)2 demonstrated reversible storage capacities up to 5.9 wt% H2. Further characterisation experiments are required to decipher the role of the Ti-dopant in these systems in both films and in the bulk.

Bidirectional regulation of mitochondrial gene expression during developmental neuroplasticity of visual cortex.

The first several months of life are a critical period for neuronal plasticity in the visual cortex, during which anatomical and physiological development depends upon visual experience. Rearing in darkness slows the time course of this critical period, such that at 5 weeks normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic. This study reports the identification of a subset of mitochondrial genes that are regulated in this manner. Opponent patterns of bidirectional expression were found: several genes (ATPase 6, cytochrome b, NADH dehydrogenase subunits 4 and 2) showed elevation in normal cats at 5 weeks and in dark-reared cats at 20 weeks ("plasticity" genes); others (NADH dehydrogenase subunits 3 and 5) showed the opposite ("anti-plasticity" genes). These findings add a new dimension to the growing evidence that changes in mitochondrial gene expression are involved in the neuroplastic response.

Rpm2p: separate domains promote tRNA and Rpm1r maturation in Saccharomyces cerevisiae mitochondria.

Rpm2p is a protein subunit of yeast mitochondrial RNase P and is also required for the maturation of Rpm1r, the mitochondrially-encoded RNA subunit of the enzyme. Previous work demonstrated that an insertional disruption of RPM2, which produces the C-terminally truncated protein Rpm2-DeltaCp, supports growth on glucose but cells lose some or all of their mitochondrial genome and become petite. These petites, even if they retain the RPM1 locus, lose their ability to process the 5'-ends of mitochondrial tRNA. We report here that if strains containing the truncated RPM2 allele are created and maintained on respiratory carbon sources they have wild-type mitochondrial genomes, and a significant portion of tRNA transcripts are processed. In contrast, precursor Rpm1r transcripts accumulate and mature Rpm1r is not made. These data show that one function of the deleted C-terminal region is in the maturation of Rpm1r, and that this region and mature Rpm1r are not absolutely required for RNase P activity. Finally, we demonstrate that full activity can be restored if the N-terminal and C-terminal domains of Rpm2p are supplied in trans.

Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae.

Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Rpm2, the protein subunit of mitochondrial RNase P in Saccharomyces cerevisiae, also has a role in the translation of mitochondrially encoded subunits of cytochrome c oxidase.

RPM2 is a Saccharomyces cerevisiae nuclear gene that encodes the protein subunit of mitochondrial RNase P and has an unknown function essential for fermentative growth. Cells lacking mitochondrial RNase P cannot respire and accumulate lesions in their mitochondrial DNA. The effects of a new RPM2 allele, rpm2-100, reveal a novel function of RPM2 in mitochondrial biogenesis. Cells with rpm2-100 as their only source of Rpm2p have correctly processed mitochondrial tRNAs but are still respiratory deficient. Mitochondrial mRNA and rRNA levels are reduced in rpm2-100 cells compared to wild type. The general reduction in mRNA is not reflected in a similar reduction in mitochondrial protein synthesis. Incorporation of labeled precursors into mitochondrially encoded Atp6, Atp8, Atp9, and Cytb protein was enhanced in the mutant relative to wild type, while incorporation into Cox1p, Cox2p, Cox3p, and Var1p was reduced. Pulse-chase analysis of mitochondrial translation revealed decreased rates of translation of COX1, COX2, and COX3 mRNAs. This decrease leads to low steady-state levels of Cox1p, Cox2p, and Cox3p, loss of visible spectra of aa(3) cytochromes, and low cytochrome c oxidase activity in mutant mitochondria. Thus, RPM2 has a previously unrecognized role in mitochondrial biogenesis, in addition to its role as a subunit of mitochondrial RNase P. Moreover, there is a synthetic lethal interaction between the disruption of this novel respiratory function and the loss of wild-type mtDNA. This synthetic interaction explains why a complete deletion of RPM2 is lethal.

Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis.

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Combined liver-kidney transplantation for primary hyperoxaluria type 1 in young children.

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is a rare condition in which deficiency of the liver enzyme alanine:glyoxylate aminotransferase leads to renal failure and systemic oxalosis. Combined liver-kidney transplantation (LKT) is recommended for end-stage renal failure (ESRF) in adults, but management of infants and young children is controversial. We retrospectively reviewed six children who underwent LKT for PH1. METHODS: The median age at diagnosis was 1.8 years (range 3 weeks to 7 years). Two children presented with severe infantile oxalosis at 3 and 9 weeks, five patients had ESRF with nephrocalcinosis and systemic oxalosis, (median duration of dialysis 1.3 years), and one had progressive chronic renal failure. Four children underwent combined LKT, one child staged liver then kidney, and one infant had an isolated liver transplant. The median age at transplantation was 8.9 years (range 1.7-15 years). RESULTS: Overall patient survival was four out of six. The two infants with PH1 and severe systemic oxalosis died (2 and 3 weeks post-transplant) due to cardiovascular oxalosis and sepsis. The other four children are well at median follow-up of 10 months (range 6 months to 7.4 years). No child developed hepatic rejection and all have normal liver function. Renal rejection occurred in three patients. Despite maximal medical management, oxalate deposits recurred in all renal grafts, contributing to graft loss in one (one of the infants who died), and significant dysfunction requiring haemodialysis post-transplant for 6 months. CONCLUSIONS: LKT is effective therapy for primary oxalosis with ESRF but has a high morbidity and mortality rate in children who present in infancy with nephrocalcinosis and systemic oxalosis. We feel that earlier LKT, or pre-emptive liver transplantation, may be a better therapeutic strategy to improve the outlook for these patients.

Splicing before import - an intein in a mitochondrially targeted preprotein folds and is catalytically active in the cytoplasm in vivo.

Nuclear-encoded mitochondrial proteins are cytoplasmically synthesized and imported into the organelle. The intein-containing RecA protein of Mycobacterium tuberculosis, with or without the CoxIVp mitochondrial targeting signal (MTS), was used to determine where a protein targeted to mitochondria folds and becomes catalytically active. Analysis of fractions from Saccharomyces cerevisiae cells expressing RecA without the MTS revealed that RecA and intein proteins remained cytoplasmic. With the MTS, most of RecA was directed to mitochondria, while most of the intein remained in the cytoplasm. The intein therefore folds into a catalytically active state in the cytoplasm prior to RecA import into mitochondria.

Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2.

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.

Collision in space.

On June 25, 1997, the Russian supply spacecraft Progress 234 collided with the Mir space station, rupturing Mir's pressure hull, throwing it into an uncontrolled attitude drift, and nearly forcing evacuation of the station. Like many high-profile accidents, this collision was the consequence of a chain of events leading to the final piloting errors that were its immediate cause. The discussion in this article does not resolve the relative contributions of the actions and decisions in this chain. Neither does it suggest corrective measures, many of which are straightforward and have already been implemented by the National Aeronautics and Space Administration (NASA) and the Russian Space Agency. Rather, its purpose is to identify the human factors that played a pervasive role in the incident. Workplace stress, fatigue, and sleep deprivation were identified by NASA as contributory factors in the Mir-Progress collision (Culbertson, 1997; NASA, forthcoming), but other contributing factors, such as requiring crew to perform difficult tasks for which their training is not current, could potentially become important factors in future situations.

Rotation and direction judgment from visual images head-slaved in two and three degrees-of-freedom.

The contribution to spatial awareness of adding a roll degree-of-freedom (DOF) to telepresence camera platform yaw and pitch was examined in an experiment where subjects judged direction and rotation of stationary target markers in a remote scene. Subjects viewed the scene via head-slaved camera images in a head-mounted display. Elimination of the roll DOF affected rotation judgment, but only at extreme yaw and pitch combinations, and did not affect azimuth and elevation judgement. Systematic azimuth overshoot occurred regardless of roll condition. Observed rotation misjudgments are explained by kinematic models for eye-head direction of gaze.

Localization of a time-delayed, monocular virtual object superimposed on a real environment.

Observers adjusted a pointer to match the depicted distance of a monocular virtual object viewed in a see-through, head-mounted display. Distance information was available through motion parallax produced as the observers rocked side to side. The apparent stability of the virtual object was impaired by a time delay between the observers' head motions and the corresponding change in the object position on the display. Localizations were made for four time delays (31 ms, 64 ms, 131 ms, and 197 ms) and three depicted distances (75 cm, 95 cm, and 113 cm). The errors in localizations increased systematically with time delay and depicted distance. A model of the results shows that the judgment error and lateral projected position of the virtual object are each linearly related to time delay.

Yeast proteins related to the p40/laminin receptor precursor are required for 20S ribosomal RNA processing and the maturation of 40S ribosomal subunits.

Numerous studies have linked the overexpression of the Mr 37,000 laminin receptor precursor (37-LRP) to tumor cell growth and proliferation. The role of this protein in carcinogenesis is generally considered in the context of its putative role as a precursor for the Mr 67,000 high-affinity laminin receptor. Recent studies have shown that 37-LRP, also termed p40, is a component of the small ribosomal subunit indicating that it may be a multifunctional protein. The p40/37-LRP protein is highly conserved phylogenetically, and closely related proteins have been identified in species as evolutionarily distant as humans and the yeast, Saccharomyces cerevisiae. Yeast homologues of p40/37-LRP are encoded by a duplicated pair of genes, RPS0A and RPS0B. The Rps0 proteins are essential components of the 40S ribosomal subunit. Previous results have shown that cells disrupted in either of the RPS0 genes have a reduction in growth rate and reduced amounts of 40S ribosomal subunits relative to wild-type cells. Here, we show that the Rps0 proteins are required for the processing of the 20S rRNA-precursor to mature 18S rRNA, a late step in the maturation of 40S ribosomal subunits. Immature subunits that are depleted of Rps0 protein that contain the 20S rRNA precursor are preferentially excluded from polysomes, which indicates that their activity in protein synthesis is dramatically reduced relative to mature 40S ribosomal subunits. These data demonstrate that the assembly of Rps0 proteins into immature 40S subunits and the subsequent processing of 20S rRNA represent critical steps in defining the translational capacity of yeast cells. If the function of these yeast proteins is representative of other members of the p40/37-LRP family of proteins, then the role of these proteins as key components of the protein synthetic machinery should also be considered as a basis for the linkage between the their overexpression and tumor cell growth and proliferation.