Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.

Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert alpha-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.

Imaging of pediatric pathology during the Iraq and Afghanistan conflicts.

United States Armed Forces radiologists deployed to Afghanistan and Iraq in modern military conflicts may encounter pediatric patients as a casualty of war or when providing humanitarian assistance to the indigenous population. Pediatric patients account for 4-7% of admissions at U.S. military hospitals during the Iraq and Afghanistan conflicts. It is pertinent for radiologists in the humanitarian care team to be familiar with imaging pediatric trauma patients, the pathology endemic to the local population, and delayed presentations of congenital and developmental disorders to adequately care for these patients. The radiological manifestations of various pediatric disorders seen in the setting of the Iraq and Afghanistan conflicts will be explored.

Quiescent fibroblasts exhibit high metabolic activity.

Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a "backwards" flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole.

Effects of aging on glucose and lipid metabolism in mice.

Aging is accompanied by multiple molecular changes that contribute to aging-associated pathologies, such as accumulation of cellular damage and mitochondrial dysfunction. Tissue metabolism can also change with age, in part because mitochondria are central to cellular metabolism. Moreover, the co-factor NAD+, which is reported to decline across multiple tissue types during aging, plays a central role in metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the oxidative synthesis of nucleotides, amino acids, and lipids. To further characterize how tissue metabolism changes with age, we intravenously infused [U-13C]-glucose into young and old C57BL/6J, WSB/EiJ, and Diversity Outbred mice to trace glucose fate into downstream metabolites within plasma, liver, gastrocnemius muscle, and brain tissues. We found that glucose incorporation into central carbon and amino acid metabolism was robust during healthy aging across these different strains of mice. We also observed that levels of NAD+, NADH, and the NAD+/NADH ratio were unchanged in these tissues with healthy aging. However, aging tissues, particularly brain, exhibited evidence of up-regulated fatty acid and sphingolipid metabolism reactions that regenerate NAD+ from NADH. Because mitochondrial respiration, a major source of NAD+ regeneration, is reported to decline with age, our data supports a model where NAD+-generating lipid metabolism reactions may buffer against changes in NAD+/NADH during healthy aging.

High-resolution ultrasonography in assessment of nail-related disorders.

OBJECTIVE: Disorders of the nail can pose a diagnostic challenge, and non-invasive imaging is frequently required to clarify diagnosis and delineate anatomy pre-operatively. We explored the use of high-resolution ultrasonography in the assessment of patients with nail disorders attending orthopaedic hand clinics. METHODS: A search of a university teaching hospital musculoskeletal radiology database identified 36 patients (mean age 54.2 years) where ultrasonography was used to assess nail-related disorders between April 2003 and January 2007. Clinical, surgical and histological findings were correlated in these cases with ultrasound reports. RESULTS: Ultrasound findings correlated with the provisional diagnosis in 20 (61%) of 33 patients and provided a diagnosis in 3 patients where a provisional diagnosis was unavailable. In 7 of the 13 cases where the clinical diagnosis differed from ultrasound findings, a lump originally diagnosed as cystic in origin was shown to be solid on ultrasound. Different nail pathologies showed different characteristics on ultrasonography, including differences in vascularity, echogenicity, changes in nail structure/shape and extension into the nail bed, matrix, fold or evidence of bony erosion. The ultrasound findings correlated with histological analysis and intra-operative assessment in 10 of 15 patients who underwent operative treatment. CONCLUSION: Ultrasound provides important information on the anatomy of the nail apparatus and can differentiate solid and cystic lesions. It can be used as a diagnostic tool and can therefore help in pre-operative planning of nail-related disorders. In our series ultrasound supported or improved upon the clinical diagnosis in 31 (86%) out of the 36 patients presenting with nail-related disorders.

Novel insights from a multiomics dissection of the Hayflick limit.

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-ß2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.

Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli.

Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single-locus perturbations in the context of ethanol exposure. A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory-evolved ethanol-tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli.

Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concentrations in aerobic, exponentially growing Escherichia coli with glucose, glycerol or acetate as the carbon source. The total observed intracellular metabolite pool was approximately 300 mM. A small number of metabolites dominate the metabolome on a molar basis, with glutamate being the most abundant. Metabolite concentration exceeds K(m) for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the K(m) of their consuming enzymes and all reactions are near equilibrium. This may facilitate efficient flux reversibility given thermodynamic and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data.

Independent regulation of age associated fat accumulation and longevity.

Age-dependent changes in metabolism can manifest as cellular lipid accumulation, but how this accumulation is regulated or impacts longevity is poorly understood. We find that Saccharomyces cerevisiae accumulate lipid droplets (LDs) during aging. We also find that over-expressing BNA2, the first Biosynthesis of NAD+ (kynurenine) pathway gene, reduces LD accumulation during aging and extends lifespan. Mechanistically, this LD accumulation during aging is not linked to NAD+ levels, but is anti-correlated with metabolites of the shikimate and aromatic amino acid biosynthesis (SA) pathways (upstream of BNA2), which produce tryptophan (the Bna2p substrate). We provide evidence that over-expressed BNA2 skews glycolytic flux from LDs towards the SA-BNA pathways, effectively reducing LDs. Importantly, we find that accumulation of LDs does not shorten lifespan, but does protect aged cells against stress. Our findings reveal how lipid accumulation impacts longevity, and how aging cell metabolism can be rewired to modulate lipid accumulation independently from longevity.

A possible association between statin use and improved Clostridioides difficile infection mortality in veterans.

Clostridioides difficile infection (CDI) is the most common cause of nosocomial diarrhea and places a significant burden on patients and the health care system. Statins could lead to improvements in CDI clinical response due their pleiotropic effects, including immunomodulatory and lipid-lowering effects; however, few studies have assessed this association. The primary objective of this study was to compare CDI health outcomes in statin users and non-users in a national cohort of patients. This was a retrospective cohort study of all adult CDI patients receiving care from the Veterans Health Administration from 2002 to 2014. Patients were divided into two groups based on statin exposure 90 days prior to and during their first CDI encounter. CDI health outcomes, including mortality and CDI recurrence, were compared using a propensity-score matched cohort of statin users and non-users and multivariable logistic regression. A total of 26,149 patients met study inclusion criteria, of which 173 statins-users and 173 non-users were propensity score matched. Thirty-day mortality was significantly lower among statins users with CDI (12.7%) compared to non-users (20.2%) (aOR 0.34; 95% CI 0.16-0.72). Sixty-day CDI recurrence was non-significantly lower among statin-users (9.0%) compared to non-users (16.6%) (aOR 0.68; 95% CI 0.29-1.59). In this nationally-representative study of veterans with CDI, statin use was associated with lower 30-day mortality compared to non-use. Statin use was not associated with 60-day CDI recurrence.

Analytical strategies for LC-MS-based targeted metabolomics.

Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple quadrupole mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.

Post-feeding activity of Lucilia sericata (Diptera: Calliphoridae) on common domestic indoor surfaces and its effect on development.

Developmental data of forensically important blowflies used by entomologists to estimate minimum post mortem interval (mPMI) are established under controlled laboratory conditions for various temperature ranges throughout the stages of egg, 1st-3rd instar, puparia, and adult fly emergence. However, environmental conditions may influence the patterns of development and behaviour of blowflies, potentially impacting on these established development rates. Previous studies investigating indoor colonisation have focused on the delay to oviposition, with behaviour during the post-feeding phase in this setting often overlooked. The environment in which third instar larvae disperse when searching for a pupariation site may vary drastically at both outdoor and indoor scenarios, influencing the activity and distance travelled during this phase and possibly affecting developmental rates. This study investigated the effect of eight common domestic indoor surfaces on dispersal time, distance travelled, and behaviour of post-feeding Lucilia sericata as well as any resulting variation in development. It was found that pupariation and puparia length within a pupariation medium of sawdust (often used in laboratory settings) produced comparable results with that of carpeted environments (those deemed to be 'enclosed'). Non-carpeted environments (those which were 'exposed') produced a delay to pupariation likely due to increased activity and energy expenditure in searching for pupariation sites which enabled burial. In addition, the observed speed of travel during dispersal was seen via time lapse photography to be greater within 'exposed' conditions. Larvae which dispersed upon burnt laminate flooring were observed to travel faster than in all other conditions and showed the only significant variation (P=0.04) in the day of emergence in comparison to the control condition of sawdust. This study has demonstrated that wandering phase activity is affected by the environmental surface which has potential implications for estimating both the distance travelled by dispersing larvae in indoor conditions and with further research, may be a consideration in mPMI calculations.

Common and divergent features of galactose-1-phosphate and fructose-1-phosphate toxicity in yeast.

Metabolic dysregulation leading to sugar-phosphate accumulation is toxic in organisms ranging from bacteria to humans. By comparing two models of sugar-phosphate toxicity in Saccharomyces cerevisiae, we demonstrate that toxicity occurs, at least in part, through multiple, isomer-specific mechanisms, rather than a single general mechanism.

Experimental exposure of cattle to a precise aerosolised challenge of Mycobacterium bovis: a novel model to study bovine tuberculosis.

Non-aerosol models of bovine tuberculosis are limited in reproducibility and relevance to natural cases seen in farmed animals. Therefore, there is a need for aerosol models of infection in cattle that can reproduce bovine tuberculosis as seen in natural cases of the disease. This manuscript describes a cattle tuberculosis model based on the inhalation of a precisely defined dose of Mycobacterium bovis in aerosol form, and defines those sites of M. bovis deposition following aerosol inhalation. The dissemination of bacilli and the resultant pathological change following infection is also described. Cattle aged 4-5 months, were infected with approximately 10(4) colony forming units (CFU), using a Madison chamber that had been modified to deliver aerosols to calves. In Experiment 1, calves were examined for gross pathology at post mortem (PM) examination at 93 and 132 days post-infection (PI), respectively. In Experiment 2, pairs of calves were examined for gross pathology at PM examination at 1 day PI and 7 days PI, respectively. At PM examination, samples were taken for bacteriology. Retrospective counts showed that the calves inhaled between 3 x 10(4) and 8 x 10(4)CFU of M. bovis. In Experiment 1, pathology indicative of tuberculosis and detection of M. bovis by qualitative bacteriology was found throughout the lower respiratory tract (LRT). In Experiment 2, pathology was only observed in a single site of one calf at day 7 PI. Samples positive for M. bovis by bacteriology were predominantly in the LRT. The numbers of M. bovis CFU recovered and the distributions of positive sites were greater at day 7 PI than day 1 PI. This study describes an aerosol exposure method that can deliver a defined dose of M. bovis almost exclusively to the LRT. The distribution of M. bovis and lesions indicative of tuberculosis suggests this aerosol method replicates the primary mode of tuberculosis transmission in cattle.

A cross-sectional study of the impact of physiotherapy and self directed exercise on the functional outcome of internally fixed isolated unimalleolar Weber B ankle fractures.

This study aimed to measure the functional outcome and quality of life in a group of patients with the same fracture type (unimalleolar Weber B ankle fractures) treated operatively at various time points and to explore the determinants of such outcomes. A cross-sectional retrospective population study was conducted. Validated Patient Related Outcome Measures (PROMs) and patient interviews were used. Fifty-one patients were included with a mean age of 54.9 years. Mean follow-up was 25 months (range 4-46 months). Mean functional scores were high (mean AOFAS 79.2, O&M 75.7, VAS-FA 80.5). However, 32% of patients did not classify themselves as fully recovered during interviews. Patient reported self-directed exercise had a statistically significant positive effect on self-reported patient perceptions of outcome (p=0.022) and PROMs (AOFAS p=0.01, O&M p=0.016, VAS-FA p=0.011). Formal physiotherapy rehabilitation was found to have no effect on self-reported patient perceptions (p=0.242) or PROMs (AOFAS p=0.8, O&M p=0.73, VAS-FA p=0.46). Our finding that physical activity is associated with improved outcome would suggest structured exercise programmes should be considered in place of physiotherapy to optimise patient outcomes.

Antibiotic prophylaxis in orthopaedic surgery: difficult decisions in an era of evolving antibiotic resistance.

Prophylactic antibiotics can decrease the risk of wound infection and have been routinely employed in orthopaedic surgery for decades. Despite their widespread use, questions still surround the selection of antibiotics for prophylaxis, timing and duration of administration. The health economic costs associated with wound infections are significant, and the judicious but appropriate use of antibiotics can reduce this risk. This review examines the evidence behind commonly debated topics in antibiotic prophylaxis and highlights the uses and advantages of some commonly used antibiotics. Cite this article: Bone Joint J 2016;98-B:1014-19.

Shedding of Mycobacterium bovis in the nasal mucus of cattle infected experimentally with tuberculosis by the intranasal and intratracheal routes.

Four groups of six calves were infected experimentally with either a low dose of approximately 10(4) colony-forming units (cfu) or a high dose of approximately 10(6) cfu of Mycobacterium bovis. Each dose was delivered by the intranasal and intratracheal routes. More severe disease was observed in the groups inoculated with the high dose. Visible lesions were identified in 21 of the 24 animals, all of which also gave positive skin tests and interferon-gamma (IFN-gamma) responses. Nasal shedding was detected in 15 of the 24 animals and the frequency of shedding was influenced by both the route and the dose of infection; no shedding was observed in the group infected intratracheally with the low dose. Two of the 15 confirmed shedders had no visible lesions at postmortem examination; both of these calves gave IFN-gamma responses but only one was skin test positive.

The investigation and management of suspected malignant pathological fractures: a review for the general orthopaedic surgeon.

The management of malignant pathological fractures necessitates careful diagnostic work-up, pre-operative investigation, planning and multidisciplinary input from specialists in the fields of radiology, pathology, oncology, trauma and orthopaedics. Malignant and non-malignant conditions including metabolic disorders, benign tumours and pharmacological therapies can be implicated. The majority of patients who present with suspected pathological fractures will be managed by general orthopaedic and trauma surgeons rather than specialists in orthopaedic oncology. Skeletal metastases can result in considerable morbidity and predispose to pathological fractures. With advances in the medical management of malignancy, life expectancy in cancer patients is increasing, leading to an increasing risk of skeletal metastasis and the potential for pathological fractures. Conventional modes of trauma fixation for pathological fractures may not be appropriate. The aim of this review is to outline diagnostic and management strategies for patients who present with a long bone fracture that is potentially pathological in nature.

Pathogenesis of tuberculosis in cattle.

There has been a renewed interest in the pathogenesis of bovine tuberculosis in many countries, in an attempt to understand better its transmission, to improve diagnosis and assess the potential of vaccination. This paper, which overviews current knowledge of aspects of the pathogenesis of bovine tuberculosis, draws from studies of field cases and experimental infections and highlights deficiencies in current understanding. The pathogenesis of bovine tuberculosis has not received the same level of attention as with human tuberculosis, and in many instances, the processes involved in bovine tuberculosis have been drawn from studies of human tuberculosis or from small animal models of infection. This paper however, considers the successful emulation of naturally acquired tuberculosis using experimental cattle models and identifies the complex and integrated nature of microbiological, immunological and pathological events involved. Current understanding of the initiation of infection, immune responses, and subsequent pathology, which can vary significantly in individual animals are discussed. Whilst there are aspects of M. bovis that still remain elusive to scientific investigation, further studies on the pathogenesis of bovine tuberculosis are advocated as necessary to provide a better scientific basis on which to review control and eradication strategies, which are currently less than effective in many regions.