Scanning ion conductance microscopy measurement of paracellular channel conductance in tight junctions.

Elucidation of epithelial transport across transcellular or paracellular pathways promises to advance the present understanding of ion transport and enables regulation of cell junctions critical to the cell and molecular biology of the epithelium. Here, we demonstrate a new instrumental technique, potentiometric scanning ion conductance microscopy (P-SICM), that utilizes a nanoscale pipet to differentiate paracellular and transcellular transport processes at high spatial resolution. The technique is validated for well-defined polymer membranes and then employed to study wild type and claudin-deficient mutants of Madin-Darby Canine Kidney strain II (MDCKII) cells. Paracellular permeabilities conferred by claudin-2 are captured by P-SICM which demonstrates the utility to monitor apparent conductance at subcellular levels.

Multifunctional carbon nanoelectrodes fabricated by focused ion beam milling.

We report a strategy for fabrication of sub-micron, multifunctional carbon electrodes and application of these electrodes as probes for scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM). The fabrication process utilized chemical vapor deposition of parylene, followed by thermal pyrolysis to form conductive carbon and then further deposition of parylene to form an insulation layer. To achieve well-defined electrode geometries, two methods of electrode exposure were utilized. In the first method, carbon probes were masked in polydimethylsiloxane (PDMS) to obtain a cone-shaped electrode. In the second method, the electrode area was exposed via milling with a focused ion beam (FIB) to reveal a carbon ring electrode, carbon ring/platinum disk electrode, or carbon ring/nanopore electrode. Carbon electrodes were batch fabricated (~35/batch) through the vapor deposition process and were characterized with scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), and cyclic voltammetry (CV) measurements. Additionally, Raman spectroscopy was utilized to examine the effects of Ga(+) ion implantation, a result of FIB milling. Constant-height, feedback mode SECM was performed with conical carbon electrodes and carbon ring electrodes. We demonstrate the utility of carbon ring/nanopore electrodes with SECM-SICM to simultaneously collect topography, ion current and electrochemical current images. In addition, carbon ring/nanopore electrodes were utilized in substrate generation/tip collection (SG/TC) SECM. In SG/TC SECM, localized delivery of redox molecules affords a higher resolution, than when the redox molecules are present in the bath solution. Multifunctional geometries of carbon electrode probes will find utility in electroanalytical applications, in general, and more specifically with electrochemical microscopy as discussed herein.

Analysis of biotinylated generation 4 poly(amidoamine) (PAMAM) dendrimer distribution in the rat brain and toxicity in a cellular model of the blood-brain barrier.

Dendrimers are highly customizable nanopolymers with qualities that make them ideal for drug delivery. The high binding affinity of biotin/avidin provides a useful approach to fluorescently label synthesized dendrimer-conjugates in cells and tissues. In addition, biotin may facilitate delivery of dendrimers through the blood-brain barrier (BBB) via carrier-mediated endocytosis. The purpose of this research was to: (1) measure toxicity using lactate dehydrogenase (LDH) assays of generation (G)4 biotinylated and non-biotinylated poly(amidoamine) (PAMAM) dendrimers in a co-culture model of the BBB, (2) determine distribution of dendrimers in the rat brain, kidney, and liver following systemic administration of dendrimers, and (3) conduct atomic force microscopy (AFM) on rat brain sections following systemic administration of dendrimers. LDH measurements showed that biotinylated dendrimers were toxic to cell co-culture after 48 h of treatment. Distribution studies showed evidence of biotinylated and non-biotinylated PAMAM dendrimers in brain. AFM studies showed evidence of dendrimers only in brain tissue of treated rats. These results indicate that biotinylation does not decrease toxicity associated with PAMAM dendrimers and that biotinylated PAMAM dendrimers distribute in the brain. Furthermore, this article provides evidence of nanoparticles in brain tissue following systemic administration of nanoparticles supported by both fluorescence microscopy and AFM.

Prevalence of antimicrobial resistance in fecal Escherichia coli and Enterococcus spp. isolates from beef cow-calf operations in northern California and associations with farm practices.

Antimicrobials are necessary for the treatment of bacterial infections in animals, but increased antimicrobial resistance (AMR) is becoming a concern for veterinarians and livestock producers. This cross-sectional study was conducted on cow-calf operations in northern California to assess prevalence of AMR in Escherichia coli and Enterococcus spp. shed in feces of beef cattle of different life stages, breeds, and past antimicrobial exposures and to evaluate if any significant factors could be identified that are associated with AMR status of the isolates. A total of 244 E. coli and 238 Enterococcus isolates were obtained from cow and calf fecal samples, tested for susceptibility to 19 antimicrobials, and classified as resistant or non-susceptible to the antimicrobials for which breakpoints were available. For E. coli, percent of resistant isolates by antimicrobial were as follows: ampicillin 100% (244/244), sulfadimethoxine 25.4% (62/244), trimethoprim-sulfamethoxazole 4.9% (12/244), and ceftiofur 0.4% (1/244) while percent of non-susceptible isolates by antimicrobial were: tetracycline 13.1% (32/244), and florfenicol 19.3% (47/244). For Enterococcus spp., percent of resistant isolates by antimicrobial were as follows: ampicillin 0.4% (1/238) while percent of non-susceptible isolates by antimicrobial were tetracycline 12.6% (30/238) and penicillin 1.7% (4/238). No animal level or farm level management practices, including antimicrobial exposures, were significantly associated with differences in isolate resistant or non-susceptible status for either E. coli or Enterococcus isolates. This is contrary to the suggestion that administration of antibiotics is solely responsible for development of AMR in exposed bacteria and demonstrates that there are other factors involved, either not captured in this study or not currently well understood. In addition, the overall use of antimicrobials in this cow-calf study was lower than other sectors of the livestock industry. Limited information is available on cow-calf AMR from fecal bacteria, and the results of this study serve as a reference for future studies to support a better understanding and estimation of drivers and trends for AMR in cow-calf operations.

Transport of redox probes through single pores measured by scanning electrochemical-scanning ion conductance microscopy (SECM-SICM).

We report scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM) experiments that describe transport of redox active molecules which emanate from single pores of a track-etch membrane. Experiments are performed with electrodes which consist of a thin gold layer deposited on one side of a nanopipet. Subsequent insulation of the electrode with parylene results in a hybrid electrode for SECM-SICM measurements. Electrode fabrication is straightforward and highly parallel. For image collection, ionic current measured at the nanopipet both controls the position of the electrode with respect to the membrane surface and reports the local conductance in the vicinity of the nanopipet, while faradaic current measured at the Au electrode reports the presence of redox-active molecules. Application of a transmembrane potential difference affords additional control over migration of charged species across the membrane.

Carbon electrode fabrication from pyrolyzed parylene C.

Carbon electrodes coupled with electrochemical detection have been used extensively for the investigation of biogenic amines. Herein we report the fabrication and characterization of carbonaceous electrodes prepared from pyrolyzed parylene C (PPC) films. High-aspect ratio carbonaceous microelectrodes have been prepared by masking PPC coated pipets with an insulating parylene C film. PPC thin film electrodes were characterized electrochemically, spectroscopically, and with electron microscopy. The procedures described here offer a route to fabrication of thin film carbon electrodes that can be patterned and produced in parallel. These electrodes are similar to carbon electrodes derived from pyrolyzed photoresist films but do not require spin-coating or lithography and can readily coat three-dimensional surfaces.

Applications of nanopipettes in the analytical sciences.

In this review, we describe measurements and applications of interest to the analytical community that makes use of simple nanopipettes. Fabricated by applying heat during the separation of a glass capillary, nanopipettes provide a route for nanoscale studies of ion transport and for development of chemical and biochemical sensors. When mounted on a translation stage, nanopipettes also enable unique modes of imaging and material deposition. These facets of nanopipette research, as well as some of the unique properties of nanopipettes, will be discussed.

Clinical management and outcomes for goats, sheep, and pigs hospitalized for treatment of burn injuries sustained in wildfires: 28 cases (2006, 2015, and 2018).

OBJECTIVE: To characterize injuries and describe medical management and clinical outcomes of goats, sheep, and pigs treated at a veterinary medical teaching hospital for burn injuries sustained during wildfires. ANIMALS: Goats (n = 9), sheep (12), and pigs (7) that sustained burn injuries from wildfires. PROCEDURES: Medical records were searched to identify goats, sheep, and pigs that had burn injuries associated with California wildfires in 2006, 2015, and 2018. Data regarding signalment, physical examination findings, treatments, clinical outcomes, time to discharge from the hospital, and reasons for death or euthanasia were recorded. RESULTS: The eyes, ears, nose, mouth, hooves, perineum, and ventral aspect of the abdomen were most commonly affected in both goats and sheep. In pigs, the ventral aspect of the abdomen, distal limb extremities, ears, and tail were most commonly affected. The median (range) time to discharge from the hospital for goats and pigs was 11 (3 to 90) and 85.5 (54 to 117) days, respectively. One of 9 goats, 12 of 12 sheep, and 5 of 7 pigs died or were euthanized. Laminitis and devitalization of distal limb extremities were common complications (13/28 animals) and a common reason for considering euthanasia in sheep and pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Burn injuries in small ruminants and pigs required prolonged treatment in some cases. Results suggested prognosis for survival may be more guarded for sheep and pigs with burn injuries than for goats; however, further research is needed to confirm these findings.