Leptin stimulates synaptogenesis in hippocampal neurons via KLF4 and SOCS3 inhibition of STAT3 signaling.

Normal development of neuronal connections in the hippocampus requires neurotrophic signals, including the cytokine leptin. During neonatal development, leptin induces formation and maturation of dendritic spines, the main sites of glutamatergic synapses in the hippocampal neurons. However, the molecular mechanisms for leptin-induced synaptogenesis are not entirely understood. In this study, we reveal two novel targets of leptin in developing hippocampal neurons and address their role in synaptogenesis. First target is Kruppel-Like Factor 4 (KLF4), which we identified using a genome-wide target analysis strategy. We show that leptin upregulates KLF4 in hippocampal neurons and that leptin signaling is important for KLF4 expression in vivo. Furthermore, KLF4 is required for leptin-induced synaptogenesis, as shKLF4 blocks and upregulation of KLF4 phenocopies it. We go on to show that KLF4 requires its signal transducer and activator of transcription 3 (STAT3) binding site and thus potentially blocks STAT3 activity to induce synaptogenesis. Second, we show that leptin increases the expression of suppressor of cytokine signaling 3 (SOCS3), another well-known inhibitor of STAT3, in developing hippocampal neurons. SOCS3 is also required for leptin-induced synaptogenesis and sufficient to stimulate it alone. Finally, we show that constitutively active STAT3 blocks the effects of leptin on spine formation, while the targeted knockdown of STAT3 is sufficient to induce it. Overall, our data demonstrate that leptin increases the expression of both KLF4 and SOCS3, inhibiting the activity of STAT3 in the hippocampal neurons and resulting in the enhancement of glutamatergic synaptogenesis during neonatal development.

Olfactory responses of Amblyomma maculatum to rumen fluid and other odourants that attract blood-seeking arthropods.

Amblyomma maculatum Koch (Ixodida: Ixodidae) has emerged as a significant vector of human and companion animal diseases in the U.S.A. When expanding in range, A. maculatum can be difficult to collect in the field and control on livestock. A novel method is needed to improve the field collection of A. maculatum, as well as to control their effects as ectoparasites of livestock and companion animals. The present study aimed to test the effects of known volatiles on the activation and selection choices of A. maculatum in a laboratory-based Y-tube assay and field-based assays. Although the majority of adult A. maculatum were activated to move by five of the seven semiochemicals tested, only rumen fluid significantly attracted ticks to make a selection in the Y-tube apparatus. Rumen fluid attracted the most A. maculatum in the laboratory, with 56% (84/150) making it to the rumen Y-tube arm, although the results were not replicated in semi-field experiments. These studies highlight the need for continued work to identify attractants for tick vectors that will assist field collections. These attractants could also be incorporated into management strategies that lead to prevention technologies to reduce tick burdens on cattle or in risk areas of humans.

Leptin-induced spine formation requires TrpC channels and the CaM kinase cascade in the hippocampus.

Leptin is a critical neurotrophic factor for the development of neuronal pathways and synaptogenesis in the hypothalamus. Leptin receptors are also found in other brain regions, including the hippocampus, and a postnatal surge in leptin correlates with a time of rapid growth of dendritic spines and synapses in the hippocampus. Leptin is critical for normal hippocampal dendritic spine formation as db/db mice, which lack normal leptin receptor signaling, have a reduced number of dendritic spines in vivo. Leptin also positively influences hippocampal behaviors, such as cognition, anxiety, and depression, which are critically dependent on dendritic spine number. What is not known are the signaling mechanisms by which leptin initiates spine formation. Here we show leptin induces the formation of dendritic protrusions (thin headless, stubby and mushroom shaped spines), through trafficking and activation of TrpC channels in cultured hippocampal neurons. Leptin-activation of the TrpC current is dose dependent and blocked by targeted knockdown of the leptin receptor. The nonselective TrpC channel inhibitors SKF96365 and 2-APB or targeted knockdown of TrpC1 or 3, but not TrpC5, channels also eliminate the leptin-induced current. Leptin stimulates the phosphorylation of CaMKIγ and ß-Pix within 5 min and their activation is required for leptin-induced trafficking of TrpC1 subunits to the membrane. Furthermore, we show that CaMKIγ, CaMKK, ß-Pix, Rac1, and TrpC1/3 channels are all required for both the leptin-sensitive current and leptin-induced spine formation. These results elucidate a critical pathway underlying leptin's induction of dendritic morphological changes that initiate spine and excitatory synapse formation.

Efficacies of spinosad and a combination of chlorpyrifos-methyl and deltamethrin against phosphine-resistant Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae) on wheat.

Highly phosphine-resistant populations of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) have recently been found in Oklahoma grain storage facilities. These findings necessitate development of a phosphine resistance management strategy to ensure continued effective use of phosphine. Therefore, we investigated the efficacies of two grain insecticides, namely, spinosad applied at label rate of 1 ppm and a mixture of chlorpyrifos-methyl and deltamethrin applied at label rates of 3 and 0.5 ppm, respectively, against highly phosphine-resistant R. dominica and T. castaneum. Adult mortality and progeny production suppression of spinosad- or chlorpyrifos-methyl + deltamethrin mixture-treated wheat that had been stored for 2, 84, 168, 252, and 336 d posttreatment were assessed. We found that both spinosad and chlorpyrifos-methyl + deltamethrin were effective against phosphine-resistant R. dominica and caused 83-100% mortality and also caused total progeny production suppression for all storage periods. Spinosad was not effective against phosphine-resistant T. castaneum; the highest mortality observed was only 3% for all the storage periods. Chlorpyrifos-methyl + deltamethrin was effective against phosphine-resistant T. castaneum only in treated wheat stored for 2 and 84 d, where it caused 93-99% mortality. However, chlorpyrifos-methyl + deltamethrin was effective and achieved total suppression of progeny production in T. castaneum for all the storage periods. Spinosad was not as effective as chlorpyrifos-methyl + deltamethrin mixture at suppressing progeny production of phosphine-resistant T. castaneum. These two insecticides can be used in a phosphine resistance management strategy for R. dominica and T. castaneum in the United States.

Using FPbase: The Fluorescent Protein Database.

FPbase is a database of fluorescent proteins and their characteristics and a set of online tools that facilitate searching the database and performing experiments with fluorescent probes. This chapter serves as a general reference for using and searching the database and a guide to some of the more commonly used tools including the spectra viewer, custom microscope pages, and FRET calculator. Important caveats when evaluating the data are also discussed.

The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets.

Lipid droplets (LDs) are crucial organelles for energy storage and lipid homeostasis. Autophagy of LDs is an important pathway for their catabolism, but the molecular mechanisms mediating LD degradation by selective autophagy (lipophagy) are unknown. Here we identify spartin as a receptor localizing to LDs and interacting with core autophagy machinery, and we show that spartin is required to deliver LDs to lysosomes for triglyceride mobilization. Mutations in SPART (encoding spartin) lead to Troyer syndrome, a form of complex hereditary spastic paraplegia1. Interfering with spartin function in cultured human neurons or murine brain neurons leads to LD and triglyceride accumulation. Our identification of spartin as a lipophagy receptor, thus, suggests that impaired LD turnover contributes to Troyer syndrome development.

Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo.

Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal Days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity.

Effect of route of introduction and host cultivar on the colonization, internalization, and movement of the human pathogen Escherichia coli O157:H7 in spinach.

Human pathogens can contaminate leafy produce in the field by various routes. We hypothesized that interactions between Escherichia coli O157:H7 and spinach are influenced by the route of introduction and the leaf microenvironment. E. coli O157:H7 labeled with green fluorescent protein was dropped onto spinach leaf surfaces, simulating bacteria-laden raindrops or sprinkler irrigation, and survived on the phylloplane for at least 14 days, with increasing titers and areas of colonization over time. The same strains placed into the rhizosphere by soil infiltration remained detectable on very few plants and in low numbers (10(2) to 10(6) CFU/g fresh tissue) that decreased over time. Stem puncture inoculations, simulating natural wounding, rarely resulted in colonization or multiplication. Bacteria forced into the leaf interior survived for at least 14 days in intercellular spaces but did not translocate or multiply. Three spinach cultivars with different leaf surface morphologies were compared for colonization by E. coli O157:H7 introduced by leaf drop or soil drench. After 2 weeks, cv. Bordeaux hosted very few bacteria. More bacteria were seen on cv. Space and were dispersed over an area of up to 0.3 mm2. The highest bacterial numbers were observed on cv. Tyee but were dispersed only up to 0.15 mm2, suggesting that cv. Tyee may provide protected niches or more nutrients or may promote stronger bacterial adherence. These findings suggest that the spinach phylloplane is a supportive niche for E. coli O157:H7, but no conclusive evidence was found for natural entry into the plant interior. The results are relevant for interventions aimed at minimizing produce contamination by human pathogens.

Association of Escherichia coli O157:H7 with filth flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies (Diptera: Muscidae).

The recent outbreak of Escherichia coli O157:H7 infection associated with contaminated spinach led to an investigation of the role of insects, which frequent fields of leafy greens and neighboring rangeland habitats, in produce contamination. Four leafy greens fields adjacent to cattle-occupied rangeland habitats were sampled using sweep nets and sticky traps. Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies because of their development in animal feces) were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR amplification using primers for the E. coli O157:H7-specific eae gene. Under laboratory conditions, house flies were confined on manure or agar medium containing E. coli O157:H7 tagged with green fluorescent protein (GFP) and then tested for their capacity to transfer the microbes to spinach plants. GFP-tagged bacteria were detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR. These results indicate that flies are capable of contaminating leafy greens under experimental conditions and confirm the importance of further investigation of the role of insects in contamination of fresh produce.

LDAF1 and Seipin Form a Lipid Droplet Assembly Complex.

Lipid droplets (LDs) originate from the endoplasmic reticulum (ER) to store triacylglycerol (TG) and cholesterol esters. The ER protein seipin was shown to localize to ER-LD contacts soon after LDs form, but what determines the sites of initial LD biogenesis in the ER is unknown. Here, we identify TMEM159, now re-named lipid droplet assembly factor 1 (LDAF1), as an interaction partner of seipin. Together, LDAF1 and seipin form an ∼600 kDa oligomeric complex that copurifies with TG. LDs form at LDAF1-seipin complexes, and re-localization of LDAF1 to the plasma membrane co-recruits seipin and redirects LD formation to these sites. Once LDs form, LDAF1 dissociates from seipin and moves to the LD surface. In the absence of LDAF1, LDs form only at significantly higher cellular TG concentrations. Our data suggest that the LDAF1-seipin complex is the core protein machinery that facilitates LD biogenesis and determines the sites of their formation in the ER.

Compounded Low-dose Naltrexone for the Treatment of Guttate Psoriasis: A Case Report.

The ancient autoimmune skin condition psoriasis is still ubiquitous worldwide, produces the same, often-intolerable effects noted in its earliest recorded descriptions, and remains without cure. Management options designed to resolve the itchy, scaly, weeping, erythematous, and often widespread lesions of that disorder are now available, but they vary in efficacy, most are associated with the development of severe adverse effects, and many are prohibitively expensive. In this article, we describe the successful use of a compounded formulation of oral low-dose naltrexone to manage guttate psoriasis in a 75-year-old white male patient. That therapy produced only 1 adverse effect (dry skin near the lesions on the patient's arms and legs) and was relatively inexpensive. The formulation for that preparation and comments from the patient, the pharmacist who suggested its use, the prescriber, and the compounder who prepared it are included. Many clinicians are unaware of the benefits of compounded low-dose naltrexone in treating autoimmune diseases. We hope that this case report will encourage compounding pharmacists to consider and suggest it as an alternative therapy for patients who cannot tolerate or afford manufactured medications to treat psoriasis.

Age-dependent effects of environmental enrichment on spatial reference memory in male mice.

Although environmental enrichment has been shown to improve various types of memory in young and aging mice, no study has directly compared the degree to which enrichment improves memory at different ages throughout the lifespan in male mice. Therefore, the present study investigated the effects of long-term continuous enrichment in young (3 months), middle-aged (15 months), and aged (21 months) male C57BL/6 mice. Spatial reference memory was tested in the Morris water maze. Results demonstrate that 24h/day environmental enrichment for approximately 6 weeks significantly improved spatial memory in the Morris water maze in aged males, but not in young or middle-aged males. These data also indicate that 24h exposure to complex enriched housing conditions increases the magnitude of enrichment-induced improvements in memory among aged mice relative to those previously reported by this lab and others.

Navigating challenges in the application of superresolution microscopy.

In 2014, the Nobel Prize in Chemistry was awarded to three scientists who have made groundbreaking contributions to the field of superresolution (SR) microscopy (SRM). The first commercial SR microscope came to market a decade earlier, and many other commercial options have followed. As commercialization has lowered the barrier to using SRM and the awarding of the Nobel Prize has drawn attention to these methods, biologists have begun adopting SRM to address a wide range of questions in many types of specimens. There is no shortage of reviews on the fundamental principles of SRM and the remarkable achievements made with these methods. We approach SRM from another direction: we focus on the current practical limitations and compromises that must be made when designing an SRM experiment. We provide information and resources to help biologists navigate through common pitfalls in SRM specimen preparation and optimization of image acquisition as well as errors and artifacts that may compromise the reproducibility of SRM data.

Filth Fly Activity Associated With Composted and Noncomposted Beef Cadavers and Laboratory Studies on Volatile Organic Compounds.

Commercial livestock facilities are faced with the challenge of managing large amounts of waste including manure and animal mortalities. One method of disposing of dead animals is composting. The cadavers are enveloped in carbon material that creates a barrier between the dead tissue and the surrounding environment. Dead tissue can release materials that not only contaminate the soil but also the groundwater and nearby surface water. Animal cadaver composting is designed to facilitate decomposition without the aid of carrion-feeding insects and reduce the presence of common pathogens associated with animal waste and dead tissue. The aim of this study was to evaluate insect activity associated with composted and exposed beef cadavers, specifically filth flies that can serve as mechanical vectors of important human pathogens such as E. coli 0157:H7. Greater numbers of all types of arthropods were trapped overall at the exposed animal site than the composted animal site. Most importantly, the number of filth flies was significantly lower at the composted site (P = 0.0009). Laboratory analysis of volatile organic compounds from composted and noncomposted rats indicated that known fly attractants such as dimethyl disulfide may be inhibited by the composting process. Implementing composting programs at livestock facilities could reduce the risk of flies spreading harmful pathogens to surrounding areas, including farms that grow fresh produce.

Polo-like kinase-dependent phosphorylation of the synaptonemal complex protein SYP-4 regulates double-strand break formation through a negative feedback loop.

The synaptonemal complex (SC) is an ultrastructurally conserved proteinaceous structure that holds homologous chromosomes together and is required for the stabilization of pairing interactions and the completion of crossover (CO) formation between homologs during meiosis I. Here, we identify a novel role for a central region component of the SC, SYP-4, in negatively regulating formation of recombination-initiating double-strand breaks (DSBs) via a feedback loop triggered by crossover designation in C. elegans. We found that SYP-4 is phosphorylated dependent on Polo-like kinases PLK-1/2. SYP-4 phosphorylation depends on DSB formation and crossover designation, is required for stabilizing the SC in pachytene by switching the central region of the SC from a more dynamic to a less dynamic state, and negatively regulates DSB formation. We propose a model in which Polo-like kinases recognize crossover designation and phosphorylate SYP-4 thereby stabilizing the SC and making chromosomes less permissive for further DSB formation.

Superresolution microscopy of the ß-carboxysome reveals a homogeneous matrix.

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use wide-field time-lapse and three-dimensional structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this superresolution technique is capable of distinguishing the localizations of the outer protein shell of the carboxysome and its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population-level description of the carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms have similar spatial and temporal localization, prompting a revised model of the internal arrangement of the ß-carboxysome.

Multifocus structured illumination microscopy for fast volumetric super-resolution imaging.

We here report for the first time the synergistic implementation of structured illumination microscopy (SIM) and multifocus microscopy (MFM). This imaging modality is designed to alleviate the problem of insufficient volumetric acquisition speed in super-resolution biological imaging. SIM is a wide-field super-resolution technique that allows imaging with visible light beyond the classical diffraction limit. Employing multifocus diffractive optics we obtain simultaneous wide-field 3D imaging capability in the SIM acquisition sequence, improving volumetric acquisition speed by an order of magnitude. Imaging performance is demonstrated on biological specimens.

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.

Integrin αß heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding ß-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, µm-scale measurements.

Point-of-care measured platelet inhibition correlates with a reduced risk of an adverse cardiac event after percutaneous coronary intervention: results of the GOLD (AU-Assessing Ultegra) multicenter study.

BACKGROUND: The optimal level of platelet inhibition with a glycoprotein (GP) IIb/IIIa antagonist necessary to minimize thrombotic complications in patients undergoing a percutaneous coronary intervention (PCI) is currently unknown. METHODS AND RESULTS: Five hundred patients undergoing a PCI with the planned use of a GP IIb/IIIa inhibitor had platelet inhibition measured at 10 minutes, 1 hour, 8 hours, and 24 hours after the initiation of therapy with the Ultegra Rapid Platelet Function Assay (Accumetrics). Major adverse cardiac events (MACES: composite of death, myocardial infarction, and urgent target vessel revascularization) were prospectively monitored, and the incidence correlated with the measured level of platelet function inhibition at all time points. One quarter of all patients did not achieve >/=95% inhibition 10 minutes after the bolus and experienced a significantly higher incidence of MACEs (14.4% versus 6.4%, P=0.006). Patients whose platelet function was <70% inhibited at 8 hours after the start of therapy had a MACE rate of 25% versus 8.1% for those >/=70% inhibited (P=0.009). By multivariate analysis, platelet function inhibition >/=95% at 10 minutes after the start of therapy was associated with a significant decrease in the incidence of a MACE (odds ratio 0.46, 95% CI 0.22 to 0.96, P=0.04). CONCLUSIONS: Substantial variability in the level of platelet function inhibition is achieved with GP IIb/IIIa antagonist therapy among patients undergoing PCI. The level of platelet function inhibition as measured by a point-of-care assay is an independent predictor for the risk of MACEs after PCI.