Effect of caffeine and other xanthines on liver sinusoidal endothelial cell ultrastructure.

Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as added ingredients. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 µg/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 µg/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery.

A placenta-on-a-chip model to determine the regulation of FKBPL and galectin-3 in preeclampsia.

Preeclampsia is a pregnancy-specific cardiovascular disorder, involving significant maternal endothelial dysfunction. Although inappropriate placentation due to aberrant angiogenesis, inflammation and shallow trophoblast invasion are the root causes of preeclampsia, pathogenic mechanisms are poorly understood, particularly in early pregnancy. Here, we first confirm the abnormal expression of important vascular and inflammatory proteins, FK506-binding protein-like (FKBPL) and galectin-3 (Gal-3), in human plasma and placental tissues from women with preeclampsia and normotensive controls. We then employ a three-dimensional microfluidic placental model incorporating human umbilical vein endothelial cells (HUVECs) and a first trimester trophoblast cell line (ACH-3P) to investigate FKBPL and Gal-3 signaling in inflammatory conditions. In human samples, both circulating (n = 17 controls; n = 30 preeclampsia) and placental (n ≥ 6) FKBPL and Gal-3 levels were increased in preeclampsia compared to controls (plasma: FKBPL, p < 0.0001; Gal-3, p < 0.01; placenta: FKBPL, p < 0.05; Gal-3, p < 0.01), indicative of vascular dysfunction in preeclampsia. In our placenta-on-a-chip model, we show that endothelial cells are critical for trophoblast-mediated migration and that trophoblasts effectively remodel endothelial vascular networks. Inflammatory cytokine tumour necrosis factor-α (10 ng/mL) modulates both FKBPL and Gal-3 signaling in conjunction with trophoblast migration and impairs vascular network formation (p < 0.005). Our placenta-on-a-chip recapitulates aspects of inappropriate placental development and vascular dysfunction in preeclampsia.

Protozoal food vacuoles enhance transformation in Vibrio cholerae through SOS-regulated DNA integration.

Vibrio cholerae, the bacterial pathogen responsible for the diarrheal disease cholera, resides in the aquatic environment between outbreaks. For bacteria, genetic variation by lateral gene transfer (LGT) is important for survival and adaptation. In the aquatic environment, V. cholerae is predominantly found in biofilms associated with chitinous organisms or with chitin "rain". Chitin induces competency in V. cholerae, which can lead to LGT. In the environment, V. cholerae is also subjected to predation pressure by protist. Here we investigated whether protozoal predation affected LGT using the integron as a model. Integrons facilitate the integration of mobile DNA (gene cassettes) into the bacterial chromosome. We report that protozoal predation enhances transformation of a gene cassette by as much as 405-fold. We show that oxidative radicals produced in the protozoal phagosome induces the universal SOS response, which in turn upregulates the integron-integrase, the recombinase that facilitates cassette integration. Additionally, we show that during predation, V. cholerae requires the type VI secretion system to acquire the gene cassette from Escherichia coli. These results show that protozoal predation enhances LGT thus producing genetic variants that may have increased capacity to survive grazing. Additionally, the conditions in the food vacuole may make it a "hot spot" for LGT by accumulating diverse bacteria and inducing the SOS response helping drive genetic diversification and evolution.

Reproductive plasticity and oogenesis in the queen honey bee (Apis mellifera).

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2000 eggs daily whereas workers are sterile in the queen's presence. Periodically queens stop laying: during swarming, when resources are scarce in winter, and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in the ovaries of honey bee queens that were caged inside and outside the colony. We find evidence that queens use a different combination of 'checkpoints' to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes likely use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that a key factor driving the termination of oogenesis in queens is nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but likely have adjusted physiological checkpoints to suit their highly-derived life history.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

Membrane trafficking directed by VAMP2 and syntaxin 3 in uterine epithelial cells.

Luminal uterine epithelial cells (UEC) have a surge in vesicular activity during early uterine receptivity. It has been predicted these vesicles exit the UEC via exocytosis resulting in secretion and membrane trafficking. The present study investigated the changes in SNARE proteins VAMP2 (v-SNARE) and syntaxin 3 (t-SNARE) localisation and abundance in UECs during early pregnancy in the rat. We found VAMP2 and syntaxin 3 are significantly higher on day 5.5 compared to day 1 of pregnancy. On day 5.5, VAMP2 is perinuclear and syntaxin 3 is concentrated in the apical cytoplasm compared to a cytoplasmic localisation on day 1. This change in localisation and abundance show VAMP2 and syntaxin 3 are involved in vesicular movement and membrane trafficking in UECs during early pregnancy. This study also investigated the influence of cytoskeletal disruption of microtubules and actin filaments on VAMP2 and syntaxin 3 in UECs grown in vitro, since microtubules and actin influence vesicle trafficking. As expected, this study found disruption to microtubules with colchicine and actin with cytochalasin D impacted VAMP2 and syntaxin 3 localisation. These results suggest VAMP2 and syntaxin 3 are involved in the timely trafficking of vesicular membranes to the apical surface in UECs during early pregnancy, as are of microtubules and actin.

Scoping Review of School-to-Work Transition for Youth With Intellectual Disabilities: A Practice Gap.

IMPORTANCE: Youth with intellectual disabilities (ID) have persistently poor work outcomes. Occupational therapy can support school-to-work transition but is underrepresented in transition practice. OBJECTIVE: To identify and describe interventions within the scope of occupational therapy for youth with ID who are transitioning from school to work. DATA SOURCES: MEDLINE, ERIC, PsycINFO, and CINAHL were searched, and hand searching was performed in relevant peer-reviewed journals. STUDY SELECTION AND DATA COLLECTION: Included were peer-reviewed, English-language articles published from 2004 to 2017 describing studies focused on youth with ID with no significant co-occurring physical diagnoses who were transitioning from U.S.-based school settings to paid employment. Data extraction was managed using Google Drive. Data were organized on extraction sheets by trained reviewers. The quality of each study was assessed using questions adapted from the Critical Appraisal Skills Program checklist. FINDINGS: A total of 35 articles were included, 7 of which used randomized controlled designs. All articles described interventions aligned with the Occupational Therapy Practice Framework: Domain and Process (3rd ed.), but specific mention of occupational therapy was notably absent from the literature. Interventions had little and generally low-level evidence supporting their use. CONCLUSIONS AND RELEVANCE: Significant and concerning gaps exist in the literature on school-to-work transition for youth with ID, likely impeding evidence-based practice. No included article mentioned occupational therapy or had a contributor who was an occupational therapy practitioner. Practitioners should advocate for occupational therapy's role in transition and contribute reports of occupational therapy transition services for youth with ID to the literature. WHAT THIS ARTICLE ADDS: This study demonstrates that occupational therapy is poorly represented in literature describing transition services for youth with ID. Although the articles described interventions within the occupational therapy domain, these interventions were not provided by occupational therapy practitioners and did not have a strong evidence base.

Sex and mortality in septic severe acute kidney injury.

PURPOSE: To investigate the relationship between sex and mortality and whether menopause or the intensity of renal replacement therapy (RRT) modify this relationship in patients with severe septic acute kidney injury (AKI). MATERIALS AND METHODS: Post-hoc analysis of patients with sepsis included in the Randomized Evaluation of Normal versus Augmented Level renal replacement therapy (RENAL) trial. RESULTS: Of 724 patients, 458 (63.3%) were male and 266 (36.7%) were female. The mean delivered effluent flow rate was 25.6 ±â€¯7.4 ml/kg/h (80 ±â€¯15% of prescribed dose) in males and 27.4 ±â€¯7.6 ml/kg/h (83 ±â€¯15% of prescribed dose) in females (p = .01). A total of 237 (51.7%) males and 118 (44.5%) females died within 90 days of randomization (p = .06). The adjusted hazard ratio (HR) for 90-day mortality was significantly decreased in females as compared with males (HR 0.74, 95% CI 0.57 to 0.96, p = .02). The relationship between sex and mortality was not significantly altered by menopausal status (adjusted P value for interaction 0.99) or by RRT intensity allocation (adjusted P value for interaction 0.27). CONCLUSIONS: In a cohort of patients with sepsis and severe AKI, female sex was associated with improved survival. The relationship between sex and survival was not altered by menopausal status or RRT intensity.

It is not good to talk: conversation has a fixed interference cost on attention regardless of difficulty.

It is well-documented that telephone conversations lead to impaired driving performance. Kunar et al. (Psychon Bull Rev 15:1135-1140, 2008) showed that this deficit was, in part, due to a dual-task cost of conversation on sustained visual attention. Using a multiple object tracking (MOT) task they found that the act of conversing on a hands-free telephone resulted in slower response times and increased errors compared to when participants performed the MOT task alone. The current study investigates whether the dual-task impairment of conversation on sustained attention is affected by conversation difficulty or task difficulty, and whether there was a dual-task deficit on attention when participants overheard half a conversation. Experiment 1 manipulated conversation difficulty by asking participants to discuss either easy questions or difficult questions. The results showed that there was no difference in the dual-task cost depending on conversation difficulty. Experiment 2 showed a similar dual-task deficit of attention in both an easy and a difficult visual search task. Experiments 3 and 4 showed that in contrast to work using a dot tracking and choice reaction time task (Emberson et al., Psychol Sci 21:1383-1388, 2010), there was little deficit on MOT performance of hearing half a conversation, provided people heard the conversations in their native language. The results are discussed in terms of a resource-depleted account of attentional resources showing a fixed conversational-interference cost on attention.

Higher versus Lower Continuous Renal Replacement Therapy Intensity in Critically ill Patients with Liver Dysfunction.

AIMS: To study the association between higher versus lower continuous renal replacement therapy (CRRT) intensity and mortality in critically ill patients with combined acute kidney injury and liver dysfunction. METHODS: Post-hoc analysis of patients with liver dysfunction (Sequential Organ Failure Assessment liver score ≥2 or diagnosis of liver failure/transplant) included in the Randomized Evaluation of Normal versus Augmented Level renal replacement therapy (RENAL) trial. RESULTS: Of 444 patients, 210 (47.3%) were randomized to higher intensity (effluent flow 40 mL/kg/h) and 234 (52.7%) to lower intensity (effluent flow 25 mL/kg/h) therapy. Overall, 79 and 86% of prescribed effluent flow was delivered in the higher-intensity and lower-intensity groups, respectively (p < 0.001). In total, 113 (54.1%) and 120 (51.3%) patients died in each group. On multivariable Cox regression analysis, we found no independent association between higher CRRT intensity and mortality (HR 0.93, 95% CI 0.70-1.24; p = 0.642). CONCLUSIONS: In RENAL patients with liver dysfunction, higher CRRT intensity was not associated with reduced mortality.

SaMpling Antibiotics in Renal Replacement Therapy (SMARRT): an observational pharmacokinetic study in critically ill patients.

BACKGROUND: Optimal antibiotic dosing is key to maximising patient survival, and minimising the emergence of bacterial resistance. Evidence-based antibiotic dosing guidelines for critically ill patients receiving RRT are currently not available, as RRT techniques and settings vary greatly between ICUs and even individual patients. We aim to develop a robust, evidence-based antibiotic dosing guideline for critically ill patients receiving various forms of RRT. We further aim to observe whether therapeutic antibiotic concentrations are associated with reduced 28-day mortality. METHODS/DESIGN: We designed a multi-national, observational pharmacokinetic study in critically ill patients requiring RRT. The study antibiotics will be vancomycin, linezolid, piperacillin/tazobactam and meropenem. Pharmacokinetic sampling of each patient's blood, RRT effluent and urine will take place during two separate dosing intervals. In addition, a comprehensive data set, which includes the patients' demographic and clinical parameters, as well as modality, technique and settings of RRT, will be collected. Pharmacokinetic data will be analysed using a population pharmacokinetic approach to identify covariates associated with changes in pharmacokinetic parameters in critically ill patients with AKI who are undergoing RRT for the five commonly prescribed antibiotics. DISCUSSION: Using the comprehensive data set collected, the pharmacokinetic profile of the five antibiotics will be constructed, including identification of RRT and other factors indicative of the need for altered antibiotic dosing requirements. This will enable us to develop a dosing guideline for each individual antibiotic that is likely to be relevant to any critically ill patient with acute kidney injury receiving any of the included forms of RRT. TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry ( ACTRN12613000241730 ) registered 28 February 2013.

A multicenter study on the effect of continuous hemodiafiltration intensity on antibiotic pharmacokinetics.

INTRODUCTION: Continuous renal replacement therapy (CRRT) may alter antibiotic pharmacokinetics and increase the risk of incorrect dosing. In a nested cohort within a large randomized controlled trial, we assessed the effect of higher (40 mL/kg per hour) and lower (25 mL/kg per hour) intensity CRRT on antibiotic pharmacokinetics. METHODS: We collected serial blood samples to measure ciprofloxacin, meropenem, piperacillin-tazobactam, and vancomycin levels. We calculated extracorporeal clearance (CL), systemic CL, and volume of distribution (Vd) by non-linear mixed-effects modelling. We assessed the influence of CRRT intensity and other patient factors on antibiotic pharmacokinetics. RESULTS: We studied 24 patients who provided 179 pairs of samples. Extracorporeal CL increased with higher-intensity CRRT but the increase was significant for vancomycin only (mean 28 versus 22 mL/minute; P = 0.0003). At any given prescribed CRRT effluent rate, extracorporeal CL of individual antibiotics varied widely, and the effluent-to-plasma concentration ratio decreased with increasing effluent flow. Overall, systemic CL varied to a greater extent than Vd, particularly for meropenem, piperacillin, and tazobactam, and large intra-individual differences were also observed. CRRT dose did not influence overall (systemic) CL, Vd, or half-life. The proportion of systemic CL due to CRRT varied widely and was high in some cases. CONCLUSIONS: In patients receiving CRRT, there is great variability in antibiotic pharmacokinetics, which complicates an empiric approach to dosing and suggests the need for therapeutic drug monitoring. More research is required to investigate the apparent relative decrease in clearance at higher CRRT effluent rates. TRIAL REGISTRATION: ClinicalTrials.gov NCT00221013. Registered 14 September 2005.

Loss of survival factors and activation of inflammatory cascades in brain sympathetic centers in type 1 diabetic mice.

Neuroinflammation and neurodegeneration have been observed in the brain in type 1 diabetes (T1D). However, little is known about the mediators of these effects. In T1D mice with 12- and 35-wk duration of diabetes we examined two mechanisms of neurodegeneration, loss of the neuroprotective factors insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and changes in indoleamine 2,3-dioxygenase (IDO) expression in the brain, and compared the response to age-matched controls. Furthermore, levels of matrix metalloproteinase-2 (MMP-2), nucleoside triphosphate diphosphohydrolase-1 (CD39), and ionized calcium-binding adaptor molecule 1 (Iba-1) were utilized to assess inflammatory changes in astrocytes, microglia, and blood vessels. In the diabetic hypothalamus (HYPO), we observed 20% reduction in neuronal soma diameter (P<0.05) and reduced neuronal expression of IGFBP-3 (-32%, P<0.05) and IGF-I (-15%, P<0.05) compared with controls at 35 wk. In diabetic HYPO, MMP-2 expression was increased in astrocytes (46%, P<0.01), and IDO⁺ cell density rose by (62%, P<0.05). CD39 expression dropped by 30% (P<0.05) in microglia and blood vessels. With 10 wk of systemic treatment using minocycline, an anti-inflammatory agent that crosses the blood-brain barrier, MMP-2, IDO, and CD39 levels normalized (P<0.05). Our results suggest that increased IDO and early loss of CD39⁺ protective cells lead to activation of inflammation in sympathetic centers of the CNS. As a downstream effect, the loss of the neuronal survival factors IGFBP-3 and IGF-I and the neurotoxic products of the kynurenine pathway contribute to the loss of neuronal density observed in the HYPO in T1D.

Daily protein intake and patient outcomes in severe acute kidney injury: findings of the randomized evaluation of normal versus augmented level of replacement therapy (RENAL) trial.

BACKGROUND AND AIMS: We aimed to examine the association between daily protein intake (DPI) and outcomes in patients from the Randomized Evaluation of Normal versus Augmented Level (RENAL) trial. METHODS: We analyzed the association between DPI and clinical outcomes using multivariable logistic regression, Cox proportional hazards models and time-adjusted analysis. RESULTS: During ICU stay, mean DPI was 37.6 g/day among survivors and 37.7 g/day among nonsurvivors (p = 0.96; DPI of 0.5 g/kg/day). Only 159 (10.9%) of the patients received a mean DPI of >1 g/kg. Patients with a DPI above the median had a 43.1% mortality compared with 46.1% for a DPI below the median (p = 0.25). On multivariate analysis, a lower DPI was not associated with increased odds ratios for 90-day mortality or any secondary outcomes. Cox proportional hazards models and time-adjusted analysis confirmed these findings. CONCLUSIONS: In the RENAL study, mean DPI was low. Within the confines of such low DPI, greater amounts of DPI were not independently associated with improved clinical outcomes. Video Journal Club "Cappuccino with Claudio Ronco" at www.karger.com/?doi=363175.

Calorie intake and patient outcomes in severe acute kidney injury: findings from The Randomized Evaluation of Normal vs. Augmented Level of Replacement Therapy (RENAL) study trial.

INTRODUCTION: Current practice in the delivery of caloric intake (DCI) in patients with severe acute kidney injury (AKI) receiving renal replacement therapy (RRT) is unknown. We aimed to describe calorie administration in patients enrolled in the Randomized Evaluation of Normal vs. Augmented Level of Replacement Therapy (RENAL) study and to assess the association between DCI and clinical outcomes. METHODS: We performed a secondary analysis in 1456 patients from the RENAL trial. We measured the dose and evolution of DCI during treatment and analyzed its association with major clinical outcomes using multivariable logistic regression, Cox proportional hazards models, and time adjusted models. RESULTS: Overall, mean DCI during treatment in ICU was low at only 10.9 ± 9 Kcal/kg/day for non-survivors and 11 ± 9 Kcal/kg/day for survivors. Among patients with a lower DCI (below the median) 334 of 729 (45.8%) had died at 90-days after randomization compared with 316 of 727 (43.3%) patients with a higher DCI (above the median) (P = 0.34). On multivariable logistic regression analysis, mean DCI carried an odds ratio of 0.95 (95% confidence interval (CI): 0.91-1.00; P = 0.06) per 100 Kcal increase for 90-day mortality. DCI was not associated with significant differences in renal replacement (RRT) free days, mechanical ventilation free days, ICU free days and hospital free days. These findings remained essentially unaltered after time adjusted analysis and Cox proportional hazards modeling. CONCLUSIONS: In the RENAL study, mean DCI was low. Within the limits of such low caloric intake, greater DCI was not associated with improved clinical outcomes. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT00221013.

The relationship between hypophosphataemia and outcomes during low-intensity and high-intensity continuous renal replacement therapy.

AIM: To identify risk factors for development of hypophosphataemia in patients treated with two different intensities of continuous renal replacement therapy (CRRT) and to assess the independent association of hypophosphataemia with major clinical outcomes. MATERIALS AND METHODS: We performed secondary analysis of data collected from 1441 patients during a large, multicentre randomised controlled trial of CRRT intensity. We allocated patients to two different intensities of CRRT (25mL/kg/hour vs 40 mL/kg/hour of effluent generation) and obtained daily measurement of serum phosphate levels. RESULTS: We obtained 14 115 phosphate measurements and identified 462 patients (32.1%) with hypophosphataemia, with peak incidence on Day 2 and Day 3. With lower intensity CRRT, there were 58 episodes of hypophosphataemia/1000 patient days, compared with 112 episodes/1000 patient days with higher intensity CRRT (P < 0.001). On multivariable logistic regression analysis, higher intensity CRRT, female sex, higher Acute Physiology and Chronic Health Evaluation score and hypokalaemia were independently associated with an increased odds ratio (OR) for hypophosphataemia. On multivariable models, hypophosphataemia was associated with better clinical outcomes, but when analysis was confined to patients alive at 96 hours, hypophosphataemia was not independently associated with clinical outcomes. CONCLUSIONS: Hypophosphataemia is common during CRRT and its incidence increases with greater CRRT intensity. Hypophosphataemia is not a robust independent predictor of mortality. Its greater incidence in the higher intensity CRRT arm of the Randomised Evaluation of Normal vs Augmented Level trial does not explain the lack of improved outcomes with such treatment.

Uptake of 25-hydroxyvitamin D by muscle and fat cells.

Vitamin D status, measured as serum 25-hydroxyvitamin D (25OHD) concentration, is determined by rates of input and of degradation. The half-life of 25OHD is surprisingly long for a steroid and much longer than its blood transporter, vitamin D binding protein. There is some evidence to suggest that vitamin D itself is stored in fat, whereas 25OHD concentrations are usually related to muscle-related parameters such as lean body mass and exercise. Both muscle and fat cells come from the mesenchymal cell lineage. We recently published evidence for net uptake of 25OHD into differentiated muscle cells, in a process that was megalin dependent, and speculated that this uptake might contribute to the extended half-life of 25OHD. Whether 25OHD is also taken up into cells of the adipocyte lineage is not clear. In the current study, we used the C2 muscle cell line as a source of myoblasts that were differentiated in culture to myotubes and 3T3-L1 pre-adipocytes that were differentiated into adipocytes in culture. We incubated the cells with trititated 25OHD and measured net uptake 4 and 16h afterwards. Differentiated myotubes took up labeled 25OHD in a time-dependent process to a far greater extent than myoblasts. In contrast, pre-adipocytes, but not differentiated adipocytes, accumulated labeled 25OHD in a time-dependent manner, though to a lesser extent than myotubes. Myotubes, but not myoblasts, showed megalin expression by immunohistochemistry. Pre-adipocytes, but not adipocytes, also showed expression of megalin. Since skeletal muscle consists mainly of differentiated muscle cells, while adipose tissue is mainly differentiated fat cells, it seems likely that muscle, but not fat tissue, provides a large extravascular pool through which 25OHD circulates and that this protects 25OHD from degradation. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

The neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

Negative emotional stimuli reduce contextual cueing but not response times in inefficient search.

In visual search, previous work has shown that negative stimuli narrow the focus of attention and speed reaction times (RTs). This paper investigates these two effects by first asking whether negative emotional stimuli narrow the focus of attention to reduce the learning of a display context in a contextual cueing task and, second, whether exposure to negative stimuli also reduces RTs in inefficient search tasks. In Experiment 1, participants viewed either negative or neutral images (faces or scenes) prior to a contextual cueing task. In a typical contextual cueing experiment, RTs are reduced if displays are repeated across the experiment compared with novel displays that are not repeated. The results showed that a smaller contextual cueing effect was obtained after participants viewed negative stimuli than when they viewed neutral stimuli. However, in contrast to previous work, overall search RTs were not faster after viewing negative stimuli (Experiments 2 to 4). The findings are discussed in terms of the impact of emotional content on visual processing and the ability to use scene context to help facilitate search.

Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.