Elucidating memory in the brain via single-cell transcriptomics.

Elucidating the neural mechanisms of memory in the brain is a central goal of neuroscience. Here, we discuss modern-day transcriptomics methodologies, and how they are well-poised to revolutionize our insight into memory mechanisms at unprecedented resolution and throughput. Focusing on the hippocampus and amygdala, two regions extensively examined in memory research, we show how single-cell transcriptomics technologies have been leveraged to understand the naïve state of these brain regions. Building upon this foundation, we show that these technologies can be applied to single-trial learning paradigms to comprehensively identify molecules and cells that participate in the encoding and retrieval of memory. Transcriptomics also provides an opportunity to understand the cell-type organization of the human hippocampus and amygdala, and due to conservation of these brain regions between humans and rodents, to infer behavioral and causal contributions in the human brain by leveraging rodent cell-type homologies and interventions. Ultimately, such transcriptomic technologies are poised to usher in a qualitatively novel understanding of memory in the brain.

Point-of-care fluorescence imaging predicts the presence of pathogenic bacteria in wounds: a clinical study.

OBJECTIVE: Bacteria in chronic wounds are invisible to the naked eye and can lead to delayed wound healing. Point-of-care bacterial fluorescence imaging illuminates a wound with 405nm light, triggering bacteria to produce red fluorescence and enabling real-time bacterial localisation. Prospective, single-blind clinical trials (clinicaltrials.gov #NCT02682069, #NCT03091361) were conducted to determine the positive predictive value (PPV) of this red fluorescence for detecting bacteria in chronic wounds. METHOD: Lower limb chronic wounds were imaged for bacterial fluorescence using the MolecuLight i:X imaging device. Regions positive for red fluorescence were discretely sampled using either biopsy or curettage to correlate red fluorescence signals to bacterial presence and analysed via gold standard quantitative polymerase chain reaction (qPCR) or via semi-quantitative culture analysis respectively. RESULTS: A total of 60 lower limb chronic wounds were imaged. Quantitative PCR analysis of wound tissue biopsies obtained from regions of red fluorescence yielded a PPV of 100%. Total bacterial load in these areas was ≥104 CFU/g. Semi-quantitative culture analysis of curettage scrapings from regions of red fluorescence yielded a PPV of 100%, with predominately moderate or heavy bacterial growth. There were nine distinct bacterial species detected, all common pathogens in chronic wounds. Staphylococcus aureus was the most prevalent species. CONCLUSION: Bacterial fluorescence image-guided curettage or biopsy sampling positively predicts bacterial presence in wounds at potentially harmful levels, entirely eliminating the risk of false negative sampling. Fluorescence imaging of wounds offers clinicians real-time information on a wound's bacterial burden, insight which can influence treatment decisions at the point-of care.

Respirometry increases cortisol levels in rainbow trout Oncorhynchus mykiss: implications for measurements of metabolic rate.

This study aimed to assess the extent to which chasing, handling and confining Oncorhynchus mykiss to a small respirometer chamber during respirometric experiments is stressful and affects metabolic measurements. The study observed increased cortisol levels in animals tested using a chase protocol and subsequent intermittent-flow respirometry, suggesting that this procedural treatment may stress animals.

The necessity-concerns framework: a multidimensional theory benefits from multidimensional analysis.

BACKGROUND: Patients' medication-related concerns and necessity-beliefs predict adherence. Evaluation of the potentially complex interplay of these two dimensions has been limited because of methods that reduce them to a single dimension (difference scores). PURPOSE: We use polynomial regression to assess the multidimensional effect of stroke-event survivors' medication-related concerns and necessity beliefs on their adherence to stroke-prevention medication. METHODS: Survivors (n = 600) rated their concerns, necessity beliefs, and adherence to medication. Confirmatory and exploratory polynomial regression determined the best-fitting multidimensional model. RESULTS: As posited by the necessity-concerns framework (NCF), the greatest and lowest adherence was reported by those necessity weak concerns and strong concerns/weak Necessity-Beliefs, respectively. However, as could not be assessed using a difference-score model, patients with ambivalent beliefs were less adherent than those exhibiting indifference. CONCLUSIONS: Polynomial regression allows for assessment of the multidimensional nature of the NCF. Clinicians/Researchers should be aware that concerns and necessity dimensions are not polar opposites.

Consumption and activity decline in Northern Pike (Esox lucius) during and after silver nanoparticle addition to a lake.

Silver nanoparticles (AgNPs) are antimicrobial additives in many consumer products with high potential for release into aquatic ecosystems. Though AgNPs have been shown to have negative impacts on fish in laboratory experiments, these effects are rarely observed at ecologically relevant concentrations or in situ in field settings. To evaluate ecosystem-level effects of this contaminant, AgNPs were added to a lake at the IISD Experimental Lakes Area (IISD-ELA) during 2014 and 2015. Mean total silver (TAg) concentrations in the water column were 4 µg L-1 during additions. The growth of Northern Pike (Esox lucius) declined, and their primary prey, Yellow Perch (Perca flavescens) became less abundant after AgNP exposure. Here, we used a combined contaminant-bioenergetics modeling approach to show that individual activity and both individual and population-level consumption of Northern Pike declined significantly in the lake dosed with AgNPs, which, combined with other evidence, suggests that observed declines in body size were likely a result of indirect effects (i.e., reduced prey availability). Further, we found the contaminant-bioenergetics approach was sensitive to modelled elimination rates of mercury, overestimating consumption and activity by 43% and 55%, respectively, when using the mercury elimination rate commonly used in these models versus field-derived estimates for this species. This study contributes to the growing evidence of potentially long-term negative impacts on fish from chronic exposure to environmentally relevant concentrations of AgNPs in a natural setting.

Neuronal cell types, projections, and spatial organization of the central amygdala.

The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a "bottom-up" understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.

Poor tolerability of thalidomide in end-stage oesophageal cancer.

Oesophageal cancer cachexia is a significant clinical problem, resulting in excessive morbidity and mortality. In a pilot study, 10 patients with cachexia due to advanced cancer of the oesophagus gained weight, including lean tissue, after 14-day treatment with thalidomide. Here, we present randomised placebo controlled trial data over a 6-week period to test the hypothesis that thalidomide is superior to placebo in terms of weight gain in patients with cachexia caused by oesophageal cancer. Thalidomide, 200 mg daily, or an identical placebo was given to patients with advanced oesophageal cancer. Total body weight and lean body mass were assessed in addition to drug tolerability and performance indices. Thirty-four patients were recruited. Of these, six given thalidomide and 16 given placebo completed the protocol; all withdrawals were due to adverse drug reactions or complications of disease. Thalidomide showed no benefit over placebo in participants who completed the protocol. These data suggest that thalidomide is poorly tolerated in patients with advanced cancer of the oesophagus and may not ameliorate the progression of cachexia. In the absence of hard supportive evidence, off-licence treatment with thalidomide should be used with great caution as an adjunct to nutritional support in patients with advanced cancer.

Spatially patterned excitatory neuron subtypes and projections of the claustrum.

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow anatomical region that primarily projects to the neocortex, whereas circuit-based approaches can suggest a broader claustrum region containing projections to the neocortex and other regions. Here, in the mouse, we took a bottom-up and cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum comprises two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential downstream targets. Thus, the claustrum comprises excitatory neuron subtypes with distinct molecular and projection properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.

Comparative structural and hemodynamic analysis of vascular trees.

The availability of detailed three-dimensional images of vascular trees from mammalian organs provides a wealth of essential data for understanding the processes and mechanisms of vascular patterning. Using this detailed geometric data requires the ability to compare individual representations of vascular trees in statistically meaningful ways. This article provides some comparisons of geometry and also of simulated hemodynamics, enabling the identification of similarities and differences among 10 individual specimens (5 placenta specimens and 5 lung specimens). Similar comparisons made with a series of models (starting with the simplest and increasing in complexity) enable the identification of essential features that are needed to account for the patterns and function of vascular arborization.

Facts, noise and wishful thinking: muscle protein turnover in aging and human disuse atrophy.

Surprisingly little is known about the mechanisms of muscle atrophy with aging and disuse in human beings, in contrast to rodents, from which much has been extrapolated to explain the human condition. However, this extrapolation is likely unwarranted because the time course, extent of wasting, muscle fiber involvement and alterations of muscle protein turnover are all quite different in rodent and human muscle. Furthermore, there is little evidence that static indices of protein turnover represent dynamic changes and may be misleading. With disuse there are reductions in the rate of muscle protein synthesis (MPS) large enough to explain the atrophic loss of muscle protein without a concomitant increase in proteolysis. In aging, there is no evidence that there are marked alterations in basal muscle protein turnover in healthy individuals but instead the ability to maintain muscle after feeding is compromised. This anabolic resistance is evident with physical inactivity, which exacerbates the inability to maintain muscle mass with aging. The main conclusion of this review is that in uncomplicated, non-inflammatory disuse atrophy, the facilitative change causing loss of muscle mass is a depression of MPS, exacerbated by anabolic resistance during feeding, with possible adaptive depressions, rather than increases, of muscle proteolysis.

Prey life-history and bioenergetic responses across a predation gradient.

To evaluate the importance of non-consumptive effects of predators on prey life histories under natural conditions, an index of predator abundance was developed for naturally occurring populations of a common prey fish, the yellow perch Perca flavescens, and compared to life-history variables and rates of prey energy acquisition and allocation as estimated from mass balance models. The predation index was positively related to maximum size and size at maturity in both male and female P. flavescens, but not with life span or reproductive investment. The predation index was positively related to size-adjusted specific growth rates and growth efficiencies but negatively related to model estimates of size-adjusted specific consumption and activity rates in both vulnerable (small) and invulnerable (large) size classes of P. flavescens. These observations suggest a trade-off between growth and activity rates, mediated by reduced activity in response to increasing predator densities. Lower growth rates and growth efficiencies in populations with fewer predators, despite increased consumption suggests either 1) a reduction in prey resources at lower predator densities or 2) an intrinsic cost of rapid prey growth that makes it unfavourable unless offset by a perceived threat of predation. This study provides evidence of trade-offs between growth and activity rates induced by predation risk in natural prey fish populations and illustrates how behavioural modification induced through predation can shape the life histories of prey fish species.

Cyclic stretch reduces myofibrillar protein synthesis despite increases in FAK and anabolic signalling in L6 cells.

Muscle protein synthesis is increased after exercise, but evidence is now accruing that during muscular activity it is suppressed. In life, muscles are subjected to shortening forces due to contraction, but may also be subject to stretching forces during lengthening. It would be biologically inefficient if contraction and stretch have different effects on muscle protein turnover, but little is known about the metabolic effects of stretch. To investigate this, we assessed myofibrillar and sarcoplasmic protein synthesis (MPS, SPS, respectively) by incorporation of [1-13C]proline (using gas chromatography-mass spectrometry) and anabolic signalling (by phospho-immunoblotting and kinase assays) in cultured L6 skeletal muscle cells during 30 min of cyclic stretch and over 30 min intervals for up to 120 min afterwards. SPS was unaffected, whereas MPS was suppressed by 40 +/- 0.03% during stretch, before returning to basal rates by 90-20 min afterwards. Paradoxically, stretch stimulated anabolic signalling with peak values after 2-30 min: e.g. focal adhesion kinase (FAK Tyr576/577; +28 +/- 6%), protein kinase B activity (Akt; +113 +/- 31%), p70S6K1 (ribosomal S6 kinase Thr389; 25 +/- 5%), 4E binding protein 1 (4EBP1 Thr37/46; 14 +/- 3%), eukaryotic elongation factor 2 (eEF2 Thr56; -47 +/- 4%), extracellular regulated protein kinase 1/2 (ERK1/2 Tyr202/204; +65% +/- 9%), eukaryotic initiation factor 2alpha (eIF2alpha Ser51; -20 +/- 5%, P < 0.05) and eukaryotic initiation factor 4E (eIF4E Ser209; +33 +/- 10%, P < 0.05). After stretch, except for Akt activity, stimulatory phosphorylations were sustained: e.g. FAK (+26 +/- 11%) for > or =30 min, eEF2 for > or =60 min (peak -45 +/- 4%), 4EBP1 for > or =90 min (+33 +/- 5%), and p70S6K1 remained elevated throughout (peak +64 +/- 7%). Adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was unchanged throughout. We report for the first time that acute cyclic stretch specifically suppresses MPS, despite increases in activity/phosphorylation of elements thought to increase anabolism.

Alterations of protein turnover underlying disuse atrophy in human skeletal muscle.

Unloading-induced atrophy is a relatively uncomplicated form of muscle loss, dependent almost solely on the loss of mechanical input, whereas in disease states associated with inflammation (cancer cachexia, AIDS, burns, sepsis, and uremia), there is a procatabolic hormonal and cytokine environment. It is therefore predictable that muscle loss mainly due to disuse alone would be governed by mechanisms somewhat differently from those in inflammatory states. We suggest that in vivo measurements made in human subjects using arterial-venous balance, tracer dilution, and tracer incorporation are dynamic and thus robust by comparison with static measurements of mRNA abundance and protein expression and/or phosphorylation in human muscle. In addition, measurements made with cultured cells or in animal models, all of which have often been used to infer alterations of protein turnover, appear to be different from results obtained in immobilized human muscle in vivo. In vivo measurements of human muscle protein turnover in disuse show that the primary variable that changes facilitating the loss of muscle mass is protein synthesis, which is reduced in both the postabsorptive and postprandial states; muscle proteolysis itself appears not to be elevated. The depressed postprandial protein synthetic response (a phenomenon we term "anabolic resistance") may even be accompanied by a diminished suppression of proteolysis. We therefore propose that most of the loss of muscle mass during disuse atrophy can be accounted for by a depression in the rate of protein synthesis. Thus the normal diurnal fasted-to-fed cycle of protein balance is disrupted and, by default, proteolysis becomes dominant but is not enhanced.

Regulation of leucine metabolism in man: a stable isotope study.

Leucine catabolism is regulated by either of the first two degradative steps: (reversible) transamination to the keto acid or subsequent decarboxylation. A method is described to measure rates of leucine transamination, reamination, and keto acid oxidation. The method is applied directly to humans by infusing the nonradioactive tracer, L-[15N,1-13C]leucine. Leucine transamination was found to be operating several times faster than the keto acid decarboxylation and to be of equal magnitude in adult human males under two different dietary conditions, postabsorptive and fed. These results indicate that decarboxylation, not transamination, is the rate-limiting step in normal human leucine metabolism.

Rheumatoid cachexia: a clinical perspective.

Rheumatoid cachexia is under-recognized in clinical practice. The loss of lean body tissue, which characterizes cachexia, is often compensated for by gain in body fat-so called 'cachectic obesity'-so that 85% or more RA patients have a normal BMI. Severe cachexia with loss of weight leads to increased morbidity and premature mortality but loss of muscle bulk with a normal BMI also associates with poor clinical outcomes. Increasing BMI, even into the obese range, is associated with less joint damage and reduced mortality. Measurement of body composition using DXA and other techniques is feasible but the results must be interpreted with care. Newer techniques such as whole-body MRI will help define with more confidence the mass and distribution of fat and muscle and help elucidate the relationships between body composition and outcomes. Cachexia shows little response to diet alone but progressive resistance training and anti-TNF therapies show promise in tackling this potentially disabling extra-articular feature of RA.

Tritium in fish from remote lakes in northwestern Ontario, Canada.

Tritium is most commonly generated as a by-product of nuclear reactors. As such, environmental concentrations are typically only reported near regions of interest, and background concentrations in areas unaffected by anthropogenic disturbance are not well characterized. To provide information on background levels of tritium in the natural environment, tissue-free water tritium (TFWT) and organically-bound tritium (OBT) were measured in the flesh of 106 fish collected within three lakes located at the IISD-Experimental Lakes Area (ELA) in Ontario, Canada in 2014. For the three ELA lakes studied, water tritium (HTO) activity concentration was determined to be below reliably detectable levels (0.6 Bq/L). Fish TFWT was found to be below 0.7 Bq/L, similar to the surrounding water tritium activity concentration. Fish OBT activity concentrations, at below 5 Bq/L, were also very low. Fish size was significantly related to OBT activity in Lake Whitefish and White Sucker from Lake 302, but not in other lakes. Though we observed significant differences in potential tritium exposure to humans among lakes, the levels of tritium reported here are below the Canadian natural background radiation of 1.8 mSv/y. These results provide information on background levels of tritium in freshwater fishes in Canada.

3D visualisation and quantification by microcomputed tomography of late gestational changes in the arterial and venous feto-placental vasculature of the mouse.

This study evaluates microcomputed tomography (micro-CT) as a method to obtain quantitative three-dimensional (3D) information on the arterial and venous vasculature of the mouse placenta. Surface renderings at embryonic days (E) 13.5, 15.5, and 18.5 (full term) revealed that the arterial and venous vasculature branched within the chorionic plate whereas only the arterial vasculature deeply penetrated the placenta. Umbilical vessel diameters measured by micro-CT did not significantly differ from those measured non-invasively in vivo by ultrasound biomicroscopy. Variability in umbilical diameters, and surface area and volume measurements of arterial and venous vascular trees due to experimental error was low relative to biological variability, and significant inter-litter differences within gestational ages were detected. Furthermore, umbilical vessel diameter increased significantly and incrementally to an arterial diameter of 0.631+/-0.009 mm and a venous diameter of 0.690+/-0.018 mm at E18.5. Umbilical vein diameter was 3-9% greater than the artery, and both were significantly correlated with embryonic body weight (R> or =0.96). Surface area and volume were determined for vessels greater than the minimum resolvable diameter of 0.03 mm which therefore excluded capillaries. Arterial surface area and volume were unchanged from E13.5-15.5 but then more than doubled at E18.5 (to 170+/-13 mm(2) and 7.2+/-0.8mm(3), respectively). Venous surface areas and volumes changed similarly with development although surface areas were lower than their arterial counterparts. We conclude that micro-CT has sufficient accuracy and precision to quantify late gestational changes in the 3D structure of the arterial and venous vasculature of the mouse placenta.

Spatial memory is impaired by peripubertal GnRH agonist treatment and testosterone replacement in sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) is used therapeutically to block activity within the reproductive axis through down-regulation of GnRH receptors within the pituitary gland. GnRH receptors are also expressed in non-reproductive tissues, including areas of the brain such as the hippocampus and amygdala. The impact of long-term GnRHa-treatment on hippocampus-dependent cognitive functions, such as spatial orientation, learning and memory, is not well studied, particularly when treatment encompasses a critical window of development such as puberty. The current study used an ovine model to assess spatial maze performance and memory of rams that were untreated (Controls), had both GnRH and testosterone signaling blocked (GnRHa-treated), or specifically had GnRH signaling blocked (GnRHa-treated with testosterone replacement) during the peripubertal period (8, 27 and 41 weeks of age). The results demonstrate that emotional reactivity during spatial tasks was compromised by the blockade of gonadal steroid signaling, as seen by the restorative effects of testosterone replacement, while traverse times remained unchanged during assessment of spatial orientation and learning. The blockade of GnRH signaling alone was associated with impaired retention of long-term spatial memory and this effect was not restored with the replacement of testosterone signaling. These results indicate that GnRH signaling is involved in the retention and recollection of spatial information, potentially via alterations to spatial reference memory, and that therapeutic medical treatments using chronic GnRHa may have effects on this aspect of cognitive function.

Selective activation of AMPK-PGC-1alpha or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation.

Endurance training induces a partial fast-to-slow muscle phenotype transformation and mitochondrial biogenesis but no growth. In contrast, resistance training mainly stimulates muscle protein synthesis resulting in hypertrophy. The aim of this study was to identify signaling events that may mediate the specific adaptations to these types of exercise. Isolated rat muscles were electrically stimulated with either high frequency (HFS; 6x10 repetitions of 3 s-bursts at 100 Hz to mimic resistance training) or low frequency (LFS; 3 h at 10 Hz to mimic endurance training). HFS significantly increased myofibrillar and sarcoplasmic protein synthesis 3 h after stimulation 5.3- and 2.7-fold, respectively. LFS had no significant effect on protein synthesis 3 h after stimulation but increased UCP3 mRNA 11.7-fold, whereas HFS had no significant effect on UCP3 mRNA. Only LFS increased AMPK phosphorylation significantly at Thr172 by approximately 2-fold and increased PGC-1alpha protein to 1.3 times of control. LFS had no effect on PKB phosphorylation but reduced TSC2 phosphorylation at Thr1462 and deactivated translational regulators. In contrast, HFS acutely increased phosphorylation of PKB at Ser473 5.3-fold and the phosphorylation of TSC2, mTOR, GSK-3beta at PKB-sensitive sites. HFS also caused a prolonged activation of the translational regulators p70 S6k, 4E-BP1, eIF-2B, and eEF2. These data suggest that a specific signaling response to LFS is a specific activation of the AMPK-PGC-1alpha signaling pathway which may explain some endurance training adaptations. HFS selectively activates the PKB-TSC2-mTOR cascade causing a prolonged activation of translational regulators, which is consistent with increased protein synthesis and muscle growth. We term this behavior the "AMPK-PKB switch." We hypothesize that the AMPK-PKB switch is a mechanism that partially mediates specific adaptations to endurance and resistance training, respectively.

The lesser evil: bad jobs or unemployment? A survey of mid-aged Australians.

Paid work is related to health in complex ways, posing both risks and benefits. Unemployment is associated with poor health, but some jobs may still be worse than no job at all. This research investigates that possibility. We used cross-sectional survey data from Australians aged 40-44 (N = 2497). Health measures were depression, physical health, self-rated health, and general practitioner visits. Employees were classified according to their job quality (strain, perceived job insecurity and marketability). Employee health was compared to people who were unemployed, and to people who were not in the labour force. We found that unemployed people reported worse health when compared to all employees. However, distinguishing in terms of employee's job quality revealed a more complex pattern. Poor quality jobs (characterized by insecurity, low marketability and job strain) were associated with worse health when compared to jobs with fewer or no stressors. Furthermore, people in jobs with three or more of the psychosocial stressors report health that is no better than the unemployed. In conclusion, paid work confers health benefits, but poor quality jobs which combine several psychosocial stressors could be as bad for health as being unemployed. Thus, workplace and industrial relations policies that diminish worker autonomy and security may generate short-term economic gains, but place longer-term burdens on the health of employees and the health-care system.