The CALERIE™ Genomic Data Resource.

Caloric restriction (CR) slows biological aging and prolongs healthy lifespan in model organisms. Findings from CALERIE-2™ - the first ever randomized, controlled trial of long-term CR in healthy, non-obese humans - broadly supports a similar pattern of effects in humans. To expand our understanding of the molecular pathways and biological processes underpinning CR effects in humans, we generated a series of genomic datasets from stored biospecimens collected from n=218 participants during the trial. These data constitute the first publicly-accessible genomic data resource for a randomized controlled trial of an intervention targeting the biology of aging. Datasets include whole-genome SNP genotypes, and three-timepoint-longitudinal DNA methylation, mRNA, and small RNA datasets generated from blood, skeletal muscle, and adipose tissue samples (total sample n=2327). The CALERIE Genomic Data Resource described in this article is available from the Aging Research Biobank. This mult-itissue, multi-omic, longitudinal data resource has great potential to advance translational geroscience.

Modeling flame extinction and reignition in large eddy simulations with fast chemistry.

This work seeks to support the validation of large eddy simulation models used to simulate fire suppression. The emphasis in the present study is on the prediction of flame extinction and the prevention of spurious reignition using a fast chemistry, mixing-controlled combustion model applicable to realistic fire scenarios of engineering interest. The configuration provides a buoyant, turbulent methane diffusion flame within a controlled co-flowing oxidizer. The oxidizer allows for the supply of a mixture of air and nitrogen, including conditions for which oxygen-dilution in the oxidizer leads to flame extinction. Measurements to support model validation include local profiles of thermocouple temperature and oxygen mole fraction, global combustion efficiency, and the limiting oxygen index. The present study evaluates the performance of critical-flame-temperature-based extinction and reignition models using the Fire Dynamics Simulator, an open-source fire dynamics solver. Alternate model cases are explored, each offering a unique treatment of extinction and reignition. Comparisons between simulated results and experimental measurements are used to evaluate the capability of these models to accurately describe flame extinction. Of the considered cases, those that include provisions to prevent spurious reignition show excellent agreement with measured data, whereas a baseline case lacking explicit reignition treatment fails to predict extinction.

Isoflurane causes neocortical but not hippocampal-dependent memory impairment in mice.

BACKGROUND: The aim of this study is to investigate the effect of general anaesthesia induced by isoflurane with buprenorphine on hippocampus-dependent and neocortex-dependent memory, respectively, in mice, and in addition, to compare the effects of such anaesthesia on these memory processes with the effects induced by lipopolysaccharide (LPS) administration on the same memory processes. METHODS: To assess hippocampus-dependent memory, isoflurane (for 15 min) after buprenorphine injection, or LPS 100 µg/kg (intraperitoneally) was administered 24 h before or after fear conditioning. The effect of these treatments on hippocampus-dependent memory was assessed using contextual fear-conditioning tasks at day 4. To assess neocortex-dependent memory, isoflurane anaesthesia or LPS was given 72 h after contextual fear conditioning. Neocortex-dependent memory assessment was performed at day 32. RESULTS: Unlike LPS injection, isoflurane with buprenorphine-induced anaesthesia does not impair freezing responses in hippocampus-dependent fear-conditioning memory tasks. On anterograde amnesia assessment: 49.67 ± 6.87% for the anaesthesia group and 54.5 ± 4.12% for the control group. On retrograde amnesia assessment: 47.16 ± 8.71% for the anaesthesia group and 54.5 ± 4.12% for control group; P > 0.05. Thus, neither isoflurane nor buprenorphine impair hippocampus-dependent memory. However, on the neocortex-dependent memory task, both isoflurane-induced anaesthesia and LPS-induced inflammation result in reduced freezing responses: 62.13 ± 5.80% for the anaesthesia group, 74.63 ± 5.69% for the LPS group, and 81.75 ± 3.26% for the control group; P < 0.05 compared with control group. CONCLUSION: General anaesthesia induced by isoflurane with buprenorphine may result in impairment of neocortex-dependent memory in mouse. However, general anaesthesia so induced does not impair hippocampus-dependent memory in mouse in our experimental conditions.

The PTS(Ntr) system globally regulates ATP-dependent transporters in Rhizobium leguminosarum.

Mutation of ptsP encoding EI(Ntr) of the PTS(Ntr) system in Rhizobium leguminosarum strain Rlv3841 caused a pleiotropic phenotype as observed with many bacteria. The mutant formed dry colonies and grew poorly on organic nitrogen or dicarboxylates. Most strikingly the ptsP mutant had low activity of a broad range of ATP-dependent ABC transporters. This lack of activation, which occurred post-translationally, may explain many of the pleiotropic effects. In contrast proton-coupled transport systems were not inhibited in a ptsP mutant. Regulation by PtsP also involves two copies of ptsN that code for EIIA(Ntr) , resulting in a phosphorylation cascade. As in Escherichia coli, the Rlv3841 PTS(Ntr) system also regulates K(+) homeostasis by transcriptional activation of the high-affinity ATP-dependent K(+) transporter KdpABC. This involves direct interaction of a two-component sensor regulator pair KdpDE with unphosphorylated EIIA(Ntr) . Critically, ptsP mutants, which cannot phosphorylate PtsN1 or PtsN2, had a fully activated KdpABC transporter. This is the opposite pattern from that observed with ABC transporters which apparently require phosphorylation of PtsN. These results suggest that ATP-dependent transport might be regulated via PTS(Ntr) responding to the cellular energy charge. ABC transport may be inactivated at low energy charge, conserving ATP for essential processes including K(+) homeostasis.

Peripheral orthopaedic surgery down-regulates hippocampal brain-derived neurotrophic factor and impairs remote memory in mouse.

Peripheral orthopaedic surgery induces a profound inflammatory response. This includes a substantial increase in cytokines and, especially, in the level of interleukin (IL)-1ß in the hippocampus, which has been shown to impair hippocampal-dependent memory in mice. We have employed two tests of contextual remote memory to demonstrate that the inflammatory response to surgical insult in mice also results in impairment of remote memory associated with prefrontal cortex (PFC). We have also found that, under the conditions presented in the social interaction test, peripheral orthopaedic surgery does not increase anxiety-like behaviour in our animal model. Although such surgery induces an increase in the level of IL-1ß in the hippocampus, it fails to do so in the PFC. Peripheral orthopaedic surgery also results in a reduction in the level of hippocampal brain-derived neurotrophic factor (BDNF) and this may contribute, in part, to the memory impairment found after such surgery. Our data suggest that a reduction in the level of hippocampal BDNF and an increase in the level of hippocampal IL-1ß following surgery may affect the transference of fear memory in the mouse brain.

Skeletal muscle mass recovery from atrophy in IL-6 knockout mice.

AIM: Skeletal muscle interleukin-6 (IL-6) expression is induced by continuous contraction, overload-induced hypertrophy and during muscle regeneration. The loss of IL-6 can alter skeletal muscle's growth and extracellular matrix remodelling response to overload-induced hypertrophy. Insulin-like growth factor-1 (IGF-1) gene expression and related signalling through Akt/mTOR is a critical regulator of muscle mass. The significance of IL-6 expression during the recovery from muscle atrophy is unclear. This study's purpose was to determine the effect of IL-6 loss on mouse gastrocnemius (GAS) muscle mass during recovery from hindlimb suspension (HS)-induced atrophy. METHODS: Female C57BL/6 [wild type (WT)] and IL-6 knockout (IL-6 KO) mice at 10 weeks of age were assigned to control, HS or HS followed by normal cage ambulation groups. RESULTS: GAS muscle atrophy was induced by 10 days of HS. HS induced a 20% loss of GAS mass in both WT and IL-6 KO mice. HS+7 days of recovery restored WT GAS mass to cage-control values. GAS mass from IL-6 KO mice did not return to cage-control values until HS+14 days of recovery. Both IGF-1 mRNA expression and Akt/mTOR signalling were increased in WT muscle after 1 day of recovery. In IL-6 KO muscle, IGF-1 mRNA expression was decreased and Akt/mTOR signalling was not induced after 1 day of recovery. MyoD and myogenin mRNA expression were both induced in WT muscle after 1 day of recovery, but not in IL-6 KO muscle. CONCLUSION: Muscle IL-6 expression appears important for the initial growth response during the recovery from disuse.

Mutation of GOGAT prevents pea bacteroid formation and N2 fixation by globally downregulating transport of organic nitrogen sources.

Mutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln:Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N(2) was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N(2) on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

Sensitization of the transient receptor potential vanilloid type 1 ion channel by isoflurane or sevoflurane does not result in extracellular signal-regulated kinase 1/2 activation in rat spinal dorsal horn neurons.

Clinically relevant concentrations of isoflurane or sevoflurane sensitize transient receptor potential vanilloid type 1 to several of its activators, including capsaicin. It has, moreover, been suggested these volatile general anaesthetics may augment nociceptive signalling arising from surgical procedures and thereby contribute to post-operative pain. To investigate this suggestion, we have studied intraplantar capsaicin injection-induced phosphorylation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons (which is a recognized marker of spinal nociceptive processing) in rat during isoflurane or sevoflurane anaesthesia after 60 min under anaesthesia. Control animals were anaesthetized with pentobarbital (which of itself does not activate extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons). Unilateral intraplantar capsaicin injection in control animals evoked extracellular signal-regulated kinase 1/2 phosphorylation in a group of neurons in lamina I and lamina II of the ipsilateral spinal dorsal horn in a somatotopically appropriate area. In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced extracellular signal-regulated kinase 1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipliateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetized animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetized animals reduced extracellular signal-regulated kinase 1/2 phosphorylation induced by the gases alone on both sides. These findings do not support the suggestion that isoflurane-, or sevoflurane-, induced sensitization of transient receptor potential vanilloid type 1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and consequential pain sensation.

Overload-induced skeletal muscle extracellular matrix remodelling and myofibre growth in mice lacking IL-6.

AIM: Overloading healthy skeletal muscle produces myofibre hypertrophy and extracellular matrix remodelling, and these processes are thought to be interdependent for producing muscle growth. Inflammatory cytokine interleukin-6 (IL-6) gene expression is induced in overloaded skeletal muscle, and the loss of this IL-6 induction can attenuate the hypertrophic response to overload (OV). Although the OV induction of IL-6 in skeletal muscle may be an important regulator of inflammatory processes and satellite cell proliferation, less is known about its role in the regulation of extracellular matrix remodelling. The purpose of the current study was to examine if OV-induced extracellular matrix remodelling, muscle growth, and associated gene expression were altered in mice that lack IL-6, when compared with wild-type mice. METHODS: Male C57/BL6 (WT) and C57/BL6 x IL-6(-/-) (IL-6(-/-)) mice (10 weeks of age) were assigned to either a sham control or synergist ablation OV treatments for 3, 21 or 56 days. RESULT: Plantaris muscle mass increased 59% in WT and 116% in IL-6(-/-) mice after 21 day OV. Myofibre CSA was also increased by 21 day OV in both WT and IL-6(-/-) mice. OV induced a twofold greater increase in the volume of non-contractile tissue in IL-6(-/-) muscle compared to WT. OV also induced a significantly greater accumulation of hydroxyproline and procollagen-1 mRNA in IL-6(-/-) muscle, when compared with WT muscle after 21 day OV. Transforming growth factor-beta and insulin-like growth factor-1 mRNA expression were also induced to a greater extent in IL-6(-/-) muscle when compared with WT muscle after 21 day OV. There was no effect of IL-6 loss on the induction of myogenin, and cyclin D1 mRNA expression after 3 day OV. However, MyoD mRNA expression in 3 day OV IL-6(-/-) muscle was attenuated when compared with WT OV mice. CONCLUSION: IL-6 appears to be necessary for the normal regulation of extracellular matrix remodelling during OV-induced growth.

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids.

One of the largest contributions to biologically available nitrogen comes from the reduction of N(2) to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N(2) fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N(2) fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N(2) fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis.

Characterization of a {gamma}-aminobutyric acid transport system of Rhizobium leguminosarum bv. viciae 3841.

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Diffuse infiltrative CD8+ lymphocytosis syndrome: review of the literature and report of a case.

Diffuse infiltrative lymphocytosis syndrome (DILS) is an uncommon manifestation of HIV infection characterised by persistent circulating CD8+ lymphocytosis accompanied by visceral lymphocytic infiltration. The lungs and the salivary glands are the most common organs involved. Parotid enlargement in HIV-seropositive subjects may represent HIV-associated lymphoepithelial lesions, hyperplastic lymphadenopathy, lymphoma, bacterial and viral infections, Sjögren's syndrome and DILS. Therefore confirmatory histopathological diagnosis and immunohistochemical analysis following a biopsy is essential to determine the exact nature of the salivary gland enlargement. A review of the literature of DILS and a description of a typical case of bilateral cystic lymphoepithelial lesions of the parotid glands, in an HIV-seropositive subject with DILS, is presented.

Osmotic upshift transiently inhibits uptake via ABC transporters in gram-negative bacteria.

ATP-binding cassette transporters from several rhizobia and Salmonella enterica serovar Typhimurium, but not secondarily coupled systems, were inhibited by high concentrations (100 to 500 mM) of various osmolytes, an effect reversed by the removal of the osmolyte. ABC systems were also inactivated in isolated pea bacteroids, probably due to the obligatory use of high-osmolarity isolation media. Measurement of nutrient cycling in isolated pea bacteroids is impeded by this effect.

Tuberculosis in an indigenous young white south Wales population.

Concerns about tuberculosis in young people in two linked deprived non-metropolitan communities triggered an investigation into local disease incidence and control measures. A pragmatic case-finding exercise, epidemiological enquiry and DNA genotyping were carried out to establish incidence and connections between cases over a five-year period. Of the 91 cases identified, 88 were from the white indigenous population. Approximately one third of cases were aged under thirty. Eight disease clusters were identified. Epidemiological investigation found spurious links between these, and DNA genotyping was necessary to clarify links. Seven cases and several contacts had been in prisons throughout the United Kingdom. Index cases under thirty generated more contacts, more contacts needing prophylaxis and more secondary cases than older patients. The workload resulting from tuberculosis control in younger cases was considerable. Tuberculosis remains an important problem in this population.

Independent origins and horizontal transfer of bacterial symbionts of aphids.

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.

True patient safety begins at the top. Leaders at one large health system rally around safety, avoid blame game.

Making patient safety the No. 1 priority at a hospital or clinic sounds like a easy task. It isn't. At one Oklahoma health system, an improved patient safety program is a massive effort requiring input and participation from every member of the staff. Figuring out how to convince employees that patient safety is their first priority means developing an extensive communication and education program.

The impact of managed care on nurses' workplace learning and teaching.

This paper examines the impact of managed care on the informal learning process for nurses in a major US-based health organisation. Through the analysis of focus group data we report the nurses' view of the effect recent changes have had on the nurse/patient/care relationship. Managed care, our research indicates, has transformed the learning milieus for nurses with two effects. First, nurses have seen their need for informal learning increase while the time and context for that learning has diminished. Second, the process of teaching patients and families has also been adversely affected even as managed care creates the need for more patient education. We report the analysis of the data collected at group interviews involving nurses working in both hospital and community settings of a leading US-based HMO. All interviews took place during September of 1997 at various sites in California. This study is part of a larger Social Science Research Council of Canada funded investigation into managed care in the US and Canada.

Postmenopausal hormone replacement: historical perspectives and current concerns.

Despite continuing evidence of its value, hormone replacement (HR) therapy is used by only a minority of postmenopausal women. Some of this reluctance may stem from concerns about the medicalization of menopause and the labeling of menopause as a state of failure or disease that needs to be treated. These concerns are understandable given the impact of previous efforts to apply erroneous biological models to women's physiology, often to their detriment. Some may assert that current attempts to describe menopause as a state of estrogen deficiency are as wrong as previous explanations that the backing up of menstrual blood required purging and bleeding. However, there is an abundance of research attesting to the value of HR in decreasing such menopausal symptoms as hot flashes and insomnia and in preventing chronic problems, including urogenital atrophy and osteoporosis. In addition, recent research suggests that estrogen may have positive effects on cognition. Questions about HR and breast cancer remain, and several studies have found a small increase in breast cancer among long-term estrogen users. The recent introduction of selective estrogen response modifiers may further increase the safety of HR therapy. Many women worried about breast cancer and other possible estrogenic effects of HR are seeking approaches they consider more natural to managing menopause, turning to such untested remedies as soy supplements and herbs. While awaiting rigorous trials, clinicians can help patients understand the consequences of relying on therapies that have unknown long-term safety and effectiveness.

Common but curable. Responding to symptoms of testicular cancer.

Testicular cancer is one of the most common cancers in men between the ages of 15 and 34. Symptoms of testicular cancer vary but can include scrotal nodules or swelling and a sensation of fullness or heaviness of the scrotum, which may be interpreted as pain. Four treatment options are available for testicular cancer: surgery to remove the cancer, radiation therapy, chemotherapy and bone marrow transplantation. No matter what a patient's treatment course, follow-up studies and office visits are imperative.