An integrated microfluidic and fluorescence platform for probing in vivo neuropharmacology.

Neurotechnologies and genetic tools for dissecting neural circuit functions have advanced rapidly over the past decade, although the development of complementary pharmacological method-ologies has comparatively lagged. Understanding the precise pharmacological mechanisms of neuroactive compounds is critical for advancing basic neurobiology and neuropharmacology, as well as for developing more effective treatments for neurological and neuropsychiatric disorders. However, integrating modern tools for assessing neural activity in large-scale neural networks with spatially localized drug delivery remains a major challenge. Here, we present a dual microfluidic-photometry platform that enables simultaneous intracranial drug delivery with neural dynamics monitoring in the rodent brain. The integrated platform combines a wireless, battery-free, miniaturized fluidic microsystem with optical probes, allowing for spatially and temporally specific drug delivery while recording activity-dependent fluorescence using genetically encoded calcium indicators (GECIs), neurotransmitter sensors GRAB NE and GRAB DA , and neuropeptide sensors. We demonstrate the performance this platform for investigating neuropharmacological mechanisms in vivo and characterize its efficacy in probing precise mechanistic actions of neuroactive compounds across several rapidly evolving neuroscience domains.

Closed-loop network of skin-interfaced wireless devices for quantifying vocal fatigue and providing user feedback.

Vocal fatigue is a measurable form of performance fatigue resulting from overuse of the voice and is characterized by negative vocal adaptation. Vocal dose refers to cumulative exposure of the vocal fold tissue to vibration. Professionals with high vocal demands, such as singers and teachers, are especially prone to vocal fatigue. Failure to adjust habits can lead to compensatory lapses in vocal technique and an increased risk of vocal fold injury. Quantifying and recording vocal dose to inform individuals about potential overuse is an important step toward mitigating vocal fatigue. Previous work establishes vocal dosimetry methods, that is, processes to quantify vocal fold vibration dose but with bulky, wired devices that are not amenable to continuous use during natural daily activities; these previously reported systems also provide limited mechanisms for real-time user feedback. This study introduces a soft, wireless, skin-conformal technology that gently mounts on the upper chest to capture vibratory responses associated with vocalization in a manner that is immune to ambient noises. Pairing with a separate, wirelessly linked device supports haptic feedback to the user based on quantitative thresholds in vocal usage. A machine learning-based approach enables precise vocal dosimetry from the recorded data, to support personalized, real-time quantitation and feedback. These systems have strong potential to guide healthy behaviors in vocal use.

Biodegradable, three-dimensional colorimetric fliers for environmental monitoring.

Recently reported winged microelectronic systems offer passive flight mechanisms as a dispersal strategy for purposes in environmental monitoring, population surveillance, pathogen tracking, and other applications. Initial studies indicate potential for technologies of this type, but advances in structural and responsive materials and in aerodynamically optimized geometries are necessary to improve the functionality and expand the modes of operation. Here, we introduce environmentally degradable materials as the basis of 3D fliers that allow remote, colorimetric assessments of multiple environmental parameters-pH, heavy metal concentrations, and ultraviolet exposure, along with humidity levels and temperature. Experimental and theoretical investigations of the aerodynamics of these systems reveal design considerations that include not only the geometries of the structures but also their mass distributions across a range of bioinspired designs. Preliminary field studies that rely on drones for deployment and for remote colorimetric analysis by machine learning interpretation of digital images illustrate scenarios for practical use.

Inhalation of Silver Silicate Nanoparticles Leads to Transient and Differential Microglial Activation in the Rodent Olfactory Bulb.

Engineered silver nanoparticles (AgNPs), including silver silicate nanoparticles (Ag-SiO2 NPs), are used in a wide variety of medical and consumer applications. Inhaled AgNPs have been found to translocate to the olfactory bulb (OB) after inhalation and intranasal instillation. However, the biological effects of Ag-SiO2 NPs and their potential nose-to-brain transport have not been evaluated. The present study assessed whether inhaled Ag-SiO2 NPs can elicit microglial activation in the OB. Adult Sprague-Dawley rats inhaled aerosolized Ag-SiO2 NPs at a concentration of 1 mg/ml for 6 hours. On day 0, 1, 7, and 21 post-exposure, rats were necropsied and OB were harvested. Immunohistochemistry on OB tissues were performed with anti-ionized calcium-binding adapter molecule 1 and heme oxygenase-1 as markers of microglial activation and oxidative stress, respectively. Aerosol characterization indicated Ag-SiO2 NPs were sufficiently aerosolized with moderate agglomeration and high-efficiency deposition in the nasal cavity and olfactory epithelium. Findings suggested that acute inhalation of Ag-SiO2 NPs elicited transient and differential microglial activation in the OB without significant microglial recruitment or oxidative stress. The delayed and differential pattern of microglial activation in the OB implied that inhaled Ag-SiO2 may have translocated to the central nervous system via intra-neuronal pathways.

Single transition metal atom embedded antimonene monolayers as efficient trifunctional electrocatalysts for the HER, OER and ORR: a density functional theory study.

Highly efficient, stable and cost-effective electrocatalysts for the hydrogen evolution reaction (HER), oxygen evolution reaction (OER), and oxygen reduction reaction (ORR) have been pursued for several decades. Herein, by employing density functional theory (DFT), a wide range of transition metal (TM = Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ru, Rh, Pd, Ag, Cd, Ir, Pt and Au) atoms anchored on antimonene (Sb monolayer) with a single Sb vacancy as single-atom catalysts (SACs) were investigated for their HER, OER and ORR performance. The results indicate that the defective Sb monolayer can be stable. Some TM@Sb monolayers show excellent stability and good electrical conductivity, beneficial for electron transfer during electrocatalytic reactions. The Ir@ and Pt@Sb monolayers exhibit excellent HER performance, both with about -0.01 eV of ΔG*H. The d band centre of the TM@Sb monolayer can be used to describe the binding strength between substrates and intermediates directly. The best OER electrocatalyst is the Pt@Sb monolayer, which shows an overpotential (ηOER) of 0.48 V. In contrast, the best ORR electrocatalyst is the Ag@Sb monolayer with an ηORR of 0.50 V, followed by Pd@, Rh@, Cd@ and Pt@Sb monolayers. Compared with pristine antimonene, only the noble metal atom could improve its OER and ORR performance effectively, and the Pt@Sb monolayer can be a trifunctional electrocatalyst for the HER/OER/ORR. Therefore, our calculations highlight a new type of SAC based on antimonene, which can be useful for energy conversion and storage.

Phenotypic and Genotypic Characterization of Veterinary Vibrio cincinnatiensis Isolates.

Vibrio cincinnatiensis is a halophilic species which has been found in marine and estuarine environments worldwide. The species is considered a rare pathogen for which the significance for humans is unclear. In this study, nine veterinary isolates were investigated that were obtained from domestic animals in Germany. The isolates were mostly recovered from abortion material of pigs, cattle, and horse (amnion or fetuses). One isolate was from a goose. A human clinical strain from a case of enteritis in Germany described in the literature was also included in the study. Whole-genome sequencing (WGS) of all isolates and MALDI-TOF MS (matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry) were performed to verify the species assignment. All strains were investigated for phenotypic traits including antimicrobial resistance (AMR), biochemical properties, and two virulence-associated phenotypes (hemolytic activity and resistance to human serum). WGS data and MS spectra confirmed that all veterinary isolates are closely related to the type strain V. cincinnatiensis NCTC12012. An exception was the human isolate from Germany which is related to the other isolates but could belong to another species. The isolates were similar in most biochemical phenotypes. Only one strain showed a very weak hemolytic activity against sheep erythrocytes, and serum resistance was intermediate in two strains. AMR phenotypes were more variable between the isolates. Resistances were observed against ß-lactams ampicillin and cefoxitin and against tetracycline and the sulfonamide antibiotics trimethoprim and sulfamethoxazole. Some acquired AMR genes were identified by bioinformatics analyses. WGS and MALDI-TOF MS data reveal a close relationship of the veterinary isolates and the type strain V. cincinnatiensis NCTC12012, which is a clinical human isolate. As the veterinary isolates of this study were mostly recovered from abortion material (amnions and fetuses), a zoonotic potential of the veterinary isolates seems possible.

Copper encapsulated in grass-derived phytoliths: Characterization, dissolution properties and the relation of content to soil properties.

The formation of phytoliths as a result of the precipitation of Si in many Si-rich plant species is known to encapsulate organic matter. This work aims to examine the possible encapsulation of Cu in grass phytoliths in an orange growing area, where Cu-rich fungicides have been excessively applied. Batch experiments, in combination with SEM-EDS and microscopy, were conducted for the grass-derived phytoliths and phytoliths separated from soil, thus revealing their dissolution properties, morphotypes and contents, in relation to soil properties. By measuring the Cu release accompanying the dissolution of phytoliths by different extractants, especially an Na2CO3/HNO3 solution, it was revealed that Cu was encapsulated within the silica body of the phytolith. This sink of Cu in the grass can be cycled to serve as a new Cu source in soils. Phytolith contents in the soil were up to 17.7 g kg-1 and tended to accumulate in soil depths from 0 to 20 cm. A positive correlation was found for soil phytolith and phytCu contents and may be indicative of the role of phytoliths as an enhancer of Cu accumulation in soil. It would be worth developing suitable techniques for the determination of phytCu, because common extraction/digestion methods are not suited for evaluating this Cu pool.

Tyrosine phosphatase MEG2 modulates murine development and platelet and lymphocyte activation through secretory vesicle function.

MEG2, a protein tyrosine phosphatase with a unique NH2-terminal lipid-binding domain, binds to and is modulated by the polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3. Recent data implicate MEG2 in vesicle fusion events in leukocytes. Through the genesis of Meg2-deficient mice, we demonstrate that Meg2-/- embryos manifest hemorrhages, neural tube defects including exencephaly and meningomyeloceles, cerebral infarctions, abnormal bone development, and >90% late embryonic lethality. T lymphocytes and platelets isolated from recombination activating gene 2-/- mice transplanted with Meg2-/- embryonic liver-derived hematopoietic progenitor cells showed profound defects in activation that, in T lymphocytes, was attributable to impaired interleukin 2 secretion. Ultrastructural analysis of these lymphocytes revealed near complete absence of mature secretory vesicles. Taken together, these observations suggest that MEG2-mediated modulation of secretory vesicle genesis and function plays an essential role in neural tube, vascular, and bone development as well as activation of mature platelets and lymphocytes.

Secretion of the mammalian Sec14p-like phosphoinositide-binding p45 protein.

Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. We recently cloned a 45-kDa protein from rat olfactory epithelium, which is homologous to the yeast Sec14p phosphatidylinositol (PtdIns) transfer protein and we report here that this protein binds to PtdIns(3,4,5)P3 and far weaker to less phosphorylated derivatives of PtdIns. Expression of the p45 protein in COS-1 cells resulted in accumulation of the protein in secretory vesicles and in the extracellular space. The secreted material contained PtdIns(3,4,5)P3. Our findings are the first report of a Sec14p-like protein involved in transport out of a cell and, to the best of our knowledge, inositol-containing phospholipids have not previously been detected in the extracellular space. Our findings suggest that p45 and phosphoinositides may participate in the formation of the protective mucus on nasal epithelium.

In vitro antimutagenicity of capsaicin toward heterocyclic amines in Salmonella typhimurium strain TA98.

Capsicum fruits are widely consumed as a component of the human diet. Capsaicin is the principle substance responsible for their hot, pungent taste. Heterocyclic amines (HCAs) are formed during cooking of meats and are mutagenic/carcinogenic compounds. In this study, we looked at whether capsaicin showed anti-mutagenic effects toward HCA-induced mutagenesis in Salmonella typhimurium TA98 when incubated with 0.5 mg liver S9 protein from rat, hamster and human. The HCAs used were Trp-P-2, Glu-P-1 and PhIP. Capsaicin, at non-toxic amounts of 0.25 and 0.5 micromole/plate, expressed a dose-dependent inhibition of the mutagenicity of Glu-P-1 and PhIP when they are metabolically activated by rat, hamster and human liver S9 and of Trp-P-2 when activated by rat and hamster liver S9. In contrast, capsaicin enhanced the mutagenicity of Trp-P-2 in TA98 when incubated with human liver S9. The lack of consistency in the anti-mutagenic action of capsaicin toward HCAs is puzzling and currently unresolved.

Covalent decoration of multi-walled carbon nanotubes with silica nanoparticles.

We describe a novel tunable approach for the synthesis of carbon nanotube-silica nanobead composites. The control of nanotube morphology and bead size coupled with the versatility of silica chemistry makes these structures an excellent platform for the development of biosensors, or for optical, magnetic and catalytic applications.

Control of vesicle fusion by a tyrosine phosphatase.

The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented alphaSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.

The minimal essential core of a cysteine-based protein-tyrosine phosphatase revealed by a novel 16-kDa VH1-like phosphatase, VHZ.

The smallest active protein-tyrosine phosphatase yet (only 16 kDa) is described here and given the name VHZ for VH1-like member Z because it belongs to the group of small Vaccinia virus VH1-related dual specific phosphatases exemplified by VHR, VHX, and VHY. Human VHZ is remarkably well conserved through evolution as it has species orthologs in frogs, fish, fly, and Archaea. The gene for VHZ, which we designate as DUSP25, is located on human chromosome 1q23.1 and consists of only two coding exons. VHZ is broadly expressed in tissues and cells, including resting blood lymphocytes, Jurkat T cells, HL-60, and RAMOS. In transfected cells, VHZ was located in the cytosol and in other cells also in the nucleoli. Endogenous VHZ showed a similar but more granular distribution. We show that VHZ is an active phosphatase and analyze its structure by computer modeling, which shows that in comparison with the 185-amino acid residue VHR, the 150-residue VHZ is a shortened version of VHR and contains the minimal set of secondary structure elements conserved in all known phosphatases from this class. The surface charge distribution of VHZ differs from that of VHR and is therefore unlikely to dephosphorylate mitogen-activated protein kinases. The remarkably high degree of conservation of VHZ through evolution may indicate a role in some ancient and fundamental physiological process.

Protein tyrosine phosphatases in T cell physiology.

The molecular mechanisms of signal transduction have been the focus of intense research during the last decade. In T cells, much of the work has centered on protein tyrosine kinase-mediated signaling from the TCR and cytokine receptors, while the study of protein tyrosine phosphatases has lagged behind. Nevertheless, it has now become clear that many protein tyrosine phosphatases play equally important roles in T cell physiology and that no kinase-regulated system would work without the counterbalancing participation of phosphatases. In fact, we have learned that many processes are regulated primarily on the phosphatase side. This minireview summarizes the current state-of-the art in our understanding of the regulation and biology of protein tyrosine phosphatases in T lymphocyte physiology.

VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX.

The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.

Homotypic secretory vesicle fusion induced by the protein tyrosine phosphatase MEG2 depends on polyphosphoinositides in T cells.

Sec14p homology domains are found in a large number of proteins from plants, yeast, invertebrates, and higher eukaryotes. We report that the N-terminal Sec14p homology domain of the human protein tyrosine phosphatase PTP-MEG2 binds phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in vitro and colocalizes with this lipid on secretory vesicle membranes in intact cells. Point mutations that prevented PtdIns(3,4,5)P(3) binding abrogated the capacity of PTP-MEG2 to induce homotypic secretory vesicle fusion in cells. Inhibition of cellular PtdIns(3,4,5)P(3) synthesis also rapidly reversed the effect of PTP-MEG2 on secretory vesicles. Finally, we show that several different phosphoinositide kinases colocalize with PTP-MEG2, thus allowing for local synthesis of PtdIns(3,4,5)P(3) in secretory vesicle membranes. We suggest that PTP-MEG2 through its Sec14p homology domain couples inositide phosphorylation to tyrosine dephosphorylation and the regulation of intracellular traffic of the secretory pathway in T cells.

Negative feedback regulation of the tumor suppressor PTEN by phosphoinositide-induced serine phosphorylation.

The PTEN tumor suppressor phosphatase directly counteracts the multiple functions of phosphatidylinositol 3-kinase by removing phosphate from the D3 position of inositol phospholipids. Like many lymphomas and leukemias, the Jurkat T cell line lacks PTEN protein due to frame-shift mutations in both PTEN alleles and therefore survives in long-term cell culture. We report that PTEN reintroduced into Jurkat was highly phosphorylated on serines 380 and 385 in its C terminus, particularly the former site. Phosphate was also detected at Ser(380) in PTEN in untransformed human T cells. Treatments that reduced the levels of D3-phospholipids in the cells resulted in reduced phosphorylation and accelerated degradation of PTEN. In contrast, expression of inactive PTEN-C124G or coexpression of a constitutively active protein kinase B led to increased phosphorylation and slower degradation of PTEN. These results suggest that PTEN normally is subjected to a feedback mechanism of regulation aimed at maintaining homeostatic levels of D3-phosphoinositides, which are crucial for T cell survival and activation.

Enlargement of secretory vesicles by protein tyrosine phosphatase PTP-MEG2 in rat basophilic leukemia mast cells and Jurkat T cells.

Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells.