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Vitaly Vodyanoy,1 Yasmine Daniels,2 Oleg Pustovyy,1 William A MacCrehan,2 Shin Muramoto,2 Gheorghe Stan21Department of Anatomy, Physiology and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, AL, 2Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MA, USA Background: Small metal nanoparticles obtained from animal blood were observed to be toxic to cultured cancer cells, whereas noncancerous cells were much less affected. In this work, engineered zinc and copper metal nanoparticles were produced from bulk metal rods by an underwater high-voltage discharge method. The metal nanoparticles were characterized by atomic force microscopy and X-ray photoelectron spectroscopy. The metal nanoparticles, with estimated diameters of 1 nm–2 nm, were determined to be more than 85% nonoxidized. A cell viability assay and high-resolution light microscopy showed that exposure of RG2, cultured rat brain glioma cancer cells, to the zinc and copper nanoparticles resulted in cell morphological changes, including decreased cell adherence, shrinking/rounding, nuclear condensation, and budding from cell bodies. The metal-induced cell injuries were similar to the effects of staurosporine, an active apoptotic reagent. The viability experiments conducted for zinc and copper yielded values of dissociation constants of 0.22±0.08 nmol/L (standard error [SE]) and 0.12±0.02 nmol/L (SE), respectively. The noncancerous astrocytes were not affected at the same conditions. Because metal nanoparticles were lethal to the cancer cells at sub-nanomolar concentrations, they are potentially important as nanomedicine.Purpose: Lethal concentrations of synthetic metal nanoparticles reported in the literature are a few orders of magnitude higher than the natural, blood-isolated metal nanoparticles; therefore, in this work, engineered metal nanoparticles were examined to mimic the properties of endogenous metal nanoparticles.Materials and methods: RG2, rat brain glioma cells CTX TNA2 brain rat astrocytes, obtained from the American Type Culture Collection, high-voltage discharge, atomic force microscope, X-ray photoelectron spectroscopy, high-resolution light microscopy, zeta potential measurements, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay were used in this work.Results: Engineered zinc and copper metal nanoparticles of size 1 nm–2 nm were lethal to cultured RG2 glioma cancer cells. Cell death was confirmed by MTT assay, showing that the relative viability of RG2 glioma cells is reduced in a dose-dependent manner at sub-nanomolar concentrations of the nanoparticles. The noncancerous astrocytes were not affected at the same conditions.Conclusion: The engineered and characterized zinc and copper nanoparticles are potentially significant as biomedicine.Keywords: nanoparticles, XPS, atomic force, glioma cancer cell, zinc, copper

It is well understood that odorants interact with specialized G-protein coupled receptors embedded in the ciliary membrane of olfactory sensory neurons (OSN) which initiates a voltage-generating intracellular cascade of signal transduction events that can be recorded at the epithelial level as an electroolfactogram (EOG). While the depolarizing excitatory pathway in vertebrates involving cyclic adenosine monophosphate (cAMP)-induced Na + /Ca 2+ influx and calcium-induced Cl - efflux is well established, there is evidence of potassium-associated inhibitory currents that correspond with cellular activation. While several Ca 2+ -dependent feedback mechanisms contribute to cellular deactivation which have been commonly attributed to these inhibitory currents, the frequently observed positive ionic conductance prior to excitatory depolarization have led many to suggest an additional earlier inhibitory mechanism at the receptor level that may be independent of downstream calcium influx. Due to conflicting conclusions, the role and mechanism behind Ca 2+ -independent inhibitory currents in olfactory cells is not fully understood. We investigated the functional and temporal involvement of potassium channels in odor transduction by comparing electroolfactogram (EOG) recordings in rat olfactory epithelia following ion channel inhibition and targeted activation of downstream components with or without potassium-blocking. Several K + -channel blocking agents (4-Aminopyridine, charybdotoxin, & iberiotoxin) demonstrated a diminished pre-action potential positive current that corresponded with reduced excitatory response to odor stimulation that was recovered when blockers were removed. We further assessed EOG responses in the absence of odor or with odor response enhancing zinc nanoparticles. Chemically eliciting membrane excitation in the absence of odor stimulation with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), in combination with K + -channel inhibition, further indicated potassium channel activation precedes excitatory events and is independent of cAMP-induced calcium influx. These results support previous findings of odor-activated inhibitory potassium currents that may play a functional role in subsequent G-protein activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Hagerty, Pustovyy, Globa, Vodyanoy and Singletary.)

Functional brain connectivity based on resting-state functional magnetic resonance imaging (fMRI) has been shown to be correlated with human personality and behavior. In this study, we sought to know whether capabilities and traits in dogs can be predicted from their resting-state connectivity, as in humans. We trained awake dogs to keep their head still inside a 3T MRI scanner while resting-state fMRI data was acquired. Canine behavior was characterized by an integrated behavioral score capturing their hunting, retrieving, and environmental soundness. Functional scans and behavioral measures were acquired at three different time points across detector dog training. The first time point (TP1) was prior to the dogs entering formal working detector dog training. The second time point (TP2) was soon after formal detector dog training. The third time point (TP3) was three months' post detector dog training while the dogs were engaged in a program of maintenance training for detection work. We hypothesized that the correlation between resting-state FC in the dog brain and behavior measures would significantly change during their detection training process (from TP1 to TP2) and would maintain for the subsequent several months of detection work (from TP2 to TP3). To further study the resting-state FC features that can predict the success of training, dogs at TP1 were divided into a successful group and a non-successful group. We observed a core brain network which showed relatively stable (with respect to time) patterns of interaction that were significantly stronger in successful detector dogs compared to failures and whose connectivity strength at the first time point predicted whether a given dog was eventually successful in becoming a detector dog. A second ontologically based flexible peripheral network was observed whose changes in connectivity strength with detection training tracked corresponding changes in behavior over the training program. Comparing dog and human brains, the functional connectivity between the brain stem and the frontal cortex in dogs corresponded to that between the locus coeruleus and left middle frontal gyrus in humans, suggestive of a shared mechanism for learning and retrieval of odors. Overall, the findings point toward the influence of phylogeny and ontogeny in dogs producing two dissociable functional neural networks.

Background: Intra-organic bone marrow node is predicted to be a part of the primo vascular system that plays a critical role in hematopoiesis and generation and regeneration of other cells. Two models of cell regeneration were suggested, one involving DNA synthesis and the other pertaining to DNA recycling. Objectives: The aim of this work is to extract a primo node from bone marrow, characterize its structure, understand its biochemistry and cell composition, and suggest a cell regeneration mechanism., Methods: Primo nodes were sampled from segmented halves of the rat femur. We used immunohistochemistry and high-resolution fluorescent microscopy to analyze 1200 samples obtained from 42 rats and 190 primo nodes., Results: Primo nodes in the bone marrow have an oval or round structure of about one millimeter in diameter, which is encompassed by a fine capsule, having incoming and outgoing vessels filled with the extracellular matrix and hematopoietic, mesenchymal, endothelial stem cells, as well as cells of the megakaryocyte family found in other primo nodes., Conclusion: Our findings imply that bone marrow nodes are intra-organic primo vascular nodes, and they provide ways and approaches for further investigation. Bone marrow nodes are simple to examine ex vivo in a variety of environments to assess cell regeneration mechanisms, wound healing, and organism rejuvenation and lifespan. Further research into these and other intra-organic nodes in animals and humans could lead to new regenerative medicine and longevity strategies that have yet to be discovered.

The blood and tissues of vertebrate animals and mammals contain small endogenous metal nanoparticles. These nanoparticles were observed to be composed of individual atoms of iron, copper, zinc, silver, gold, platinum, and other metals. Metal nanoparticles can bind proteins and produce proteinaceous particles called proteons. A small fraction of the entire pool of nanoparticles is usually linked with proteins to form proteons. These endogenous metal nanoparticles, along with engineered zinc and copper nanoparticles at subnanomolar levels, were shown to be lethal to cultured cancer cells. These nanoparticles appear to be elemental crystalline metal nanoparticles. It was discovered that zinc nanoparticles produce no odor response but increase the odor reaction if mixed with an odorant. Some other metal nanoparticles, including copper, silver, gold, and platinum nanoparticles, do not affect the responses to odorants. The sources of metal nanoparticles in animal blood and tissues may include dietary plants and gut microorganisms. The solid physiological and biochemical properties of metal nanoparticles reflect their importance in cell homeostasis and disease.

The role of zinc in neurobiology is rapidly expanding. Zinc is especially essential in olfactory neurobiology. Naturally occurring zinc nanoparticles were detected in olfactory and nasal respiratory epithelia and cilia in animals. The addition of these nanoparticles to a mixture of odorants, including ethyl butyrate, eugenol, and carvone, considerably increased the electrical responses of the olfactory sensory receptors. Studies of these nanoparticles by ransmission electron microscopy (TEM) and selected area electron diffraction revealed metal elemental crystalline zinc nanoparticles 2-4 nm in diameter. These particles did not contain oxidized zinc. The enhancement of the odorant responses induced by the endogenous zinc nanoparticles appears to be similar to the amplification produced by engineered zinc nanoparticles. Zinc nanoparticles produce no odor response but increase odor response if mixed with an odorant. These effects are dose-dependent and reversible. Some other metal nanoparticles, such as copper, silver, gold, and platinum, do not have the effects observed in the case of zinc nanoparticles. The olfactory enhancement was observed in young and mature mouse olfactory epithelium cultures, in the dissected olfactory epithelium of rodents, and in live conscious dogs. The physiological significance of the detected endogenous metal nanoparticles in an animal tissue has been demonstrated for the first time. Overall, our results may advance the understanding of the initial events in olfaction.

Electroacupuncture (EA) relieves visceral hypersensitivity (VH) with underlying inflammatory bowel diseases. However, the mechanism by which EA treats ileitis-induced VH is not clearly known. To assess the effects of EA on ileitis-induced VH and confirm whether EA attenuates VH through spinal PAR-2 activation and CGRP release, goats received an injection of 2,4,6-trinitro-benzenesulfonic-acid (TNBS) solution into the ileal wall. TNBS-injected goats were allocated into VH, Sham acupuncture (Sham-A) and EA groups, while goats treated with saline instead of TNBS solution were used as the control. Goats in EA group received EA at bilateral Hou-San-Li acupoints for 0.5 h at 7 days and thereafter repeated every 3 days for 6 times. Goats in the Sham-A group were inserted with needles for 0.5 h at the aforementioned acupoints without any hand manipulation and electric stimulation. Visceromotor responses to colorectal distension, an indicator of VH, were recorded by electromyography. The terminal ileum and thoracic spinal cord (T 11 ) were sampled for evaluating ileitis at days 7 and 22, and distribution and expression-levels of PAR-2, CGRP and c-Fos on day 22. TNBS-treated-goats exhibited apparent transmural-ileitis on day 7, microscopically low-grade ileitis on day 22 and VH at days 7-22. Goats of Sham-A, VH or EA group showed higher (P < 0.01) VH at days 7-22 than the Control-goats. EA-treated goats exhibited lower (P < 0.01) VH as compared with Sham-A or VH group. Immunoreactive-cells and expression-levels of spinal PAR-2, CGRP and c-Fos in the EA group were greater (P < 0.01) than those in the Control group, but less (P < 0.01) than those in Sham-A and VH groups on day 22. Downregulation of spinal PAR-2 and CGRP levels by EA attenuates the ileitis and resultant VH.

Stem cells are nurtured and regulated by a specialized microenvironment known as stem cell niche. While the functions of the niches are well defined, their structure and location remain unclear. We have identified, in rat bone marrow, the seat of hematopoietic stem cells-extensively vascularized node-like compartments that fit the requirements for stem cell niche and that we called hemmules. Hemmules are round or oval structures of about one millimeter in diameter that are surrounded by a fine capsule, have afferent and efferent vessels, are filled with the extracellular matrix and mesenchymal, hematopoietic, endothelial stem cells, and contain cells of the megakaryocyte family, which are known for homeostatic quiescence and contribution to the bone marrow environment. We propose that hemmules are the long sought hematopoietic stem cell niches and that they are prototypical of stem cell niches in other organs.

Electroacupuncture (EA) can relieve various pains. However, its mechanism in terms of the transcriptome is still not well-known. To explore the full profile of EA-induced molecular modification in the central nerve system, three twins of goats were selected for a match-paired experiment: EA stimulation (60 Hz, 30 min) and none-EA (control). Goats in the EA group showed an increased ( p < 0.05) nociceptive threshold compared with the control goats. Experimental goats were sacrificed at 4 h of the experiment, and the periaqueductal grays were harvested for RNA sequencing. As a result, 2651 differentially expressed genes (1803 up-regulated and 848 down-regulated genes) were found and enriched in 30 Kyoto Encyclopedia of Genes and Genomes pathways and 149 gene ontology terms. EA-regulated five neuropeptide genes ( proenkephalin , proopiomelanocortin , preprodynorphin , diazepam-binding inhibitor and proprotein convertase 1 inhibitor ) were validated with quantitative PCR. Furthermore, up-regulated glutamate receptors, glutamate transporters, γ-aminobutyric acid ( GABA ) receptors, GABA transporters, synaptotagmins or mitogen-activated protein kinase ( MAPK ) genes might contribute to EA-induced analgesia through regulating the glutamatergic synapse, GABAergic synapse, MAPKs, ribosome or ubiquitin-proteasome pathways. Our findings reveal a full profile of molecular modification in response to EA and provide a solid experimental framework for exploring the mechanisms underlying EA-induced analgesia., Competing Interests: The authors declare no conflict of interest.

Olfactory responses are intensely enhanced with the addition of endogenous and engineered primarily-elemental small zinc nanoparticles (NPs). With aging, oxidation of these Zn nanoparticles eliminated the observed enhancement. The design of a polyethylene glycol coating to meet storage requirements of engineered zinc nanoparticles is evaluated to achieve maximal olfactory benefit. The zinc nanoparticles were covered with 1000 g/mol or 400 g/mol molecular weight polyethylene glycol (PEG). Non-PEGylated and PEGylated zinc nanoparticles were tested by electroolfactogram with isolated rat olfactory epithelium and odorant responses evoked by the mixture of eugenol, ethyl butyrate and (±) carvone after storage at 278 K (5 oC), 303 K (30 oC) and 323 K (50 oC). The particles were analyzed by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and laser Doppler velocimetry. Our data indicate that stored ZnPEG400 nanoparticles maintain physiologically-consistent olfactory enhancement for over 300 days. These engineered Nanoparticles support future applications in olfactory research, sensitive detection, and medicine.

Electroacupuncture (EA) has been used for treating visceral hypersensitivity (VH). However, the underlying molecular mechanism remains unclear. This study was aim to testify the effect of EA on ileitis-provoked VH, and to confirm whether EA attenuates VH through Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathway in the periaqueductal gray (PAG)-the rostral ventromedial medulla (RVM)-the spinal cord dorsal horn (SCDH) axis. Methods: Goats were anesthetized and laparotomized for injecting 2,4,6-trinitro-benzene-sulfonic acid (TNBS)-ethanol solution (30mg TNBS dissolved in 40% ethanol) into the ileal wall to induce VH. EA was treated for 30min from day 7, then every 3 days for six times. VH was assessed by visceromotor response (VMR) and pain behavior response to 20, 40, 60, 80, and 100 mmHg colorectal distension pressures at day 7, 10, 13, 16, 19, and 22. The spinal cord in the eleventh thoracic vertebra and the brain were collected at day 22. The protein and mRNA levels of IL-6, JAK2, and STAT3 in the SCDH were detected with western blot and qPCR, respectively. The distribution of these substances was observed with immunohistochemistry in the ventrolateral PAG (vlPAG), RVM (mainly the nucleus raphe magnus, NRM), SCDH, the nucleus tractus solitaries (NTS) and the dorsal motor nucleus of vagi (DMV). Results: Goats administered with TNBS-ethanol solution showed diarrhea, enhanced VMR and pain behavior response, and increased IL-6, phosphorylated JAK2 and STAT3 (pJAK2 and pSTAT3) in the vlPAG, NRM, NTS and DMV, and their protein and mRNA levels in the SCDH. EA relieved diarrhea, VMR and pain behavior response, decreased IL-6, pJAK2 and pSTAT3 levels in the vlPAG, NRM, SCDH, NTS, and DMV except for pSTAT3 in the DMV, but did not affect mRNA level of these three substances in the SCDH. Conclusion: EA attenuates VH probably through inhibiting JAK2/STAT3 signaling pathway in the PAG-RVM-SCDH axis.

Since Bonghan Kim's discovery of the Bonghan system (BHS) in the 1960s, numerous reports have suggested that the system is fundamental for maintaining mammalian life. The BHS is a circulatory system independent of the blood or the lymphatic system, forms an extensive network throughout the entire mammalian body, has been reported to be the acupuncture meridian, stores distinct types of stem cells, and appears to have some roles in cancer metastasis. Despite Kim's first report having been published as early as 1962, research progress has been rather slow mainly because the system is very small and translucent, making it optically difficult to distinguish it from the hemoglobin-rich surrounding tissues. Unfortunately, Kim did not describe in detail the methods that he used for identifying and harvesting the system and the components of the system. In 2000, Kwang-Sup Soh reopened the BHS research, and since then, new and important scientific findings on the system have been reported, and many of Kim's results have been verified. In 2010, the BHS was renamed the primo vascular system. Nevertheless, good tools to properly deal with this system are still lacking. In this article, we address some of the technical difficulties involved in studying the primo vascular system and attempt to discuss potential ways to overcome those difficulties., (Copyright © 2016. Published by Elsevier B.V.)

Diffusion tensor imaging (DTI) provides us an insight into the micro-architecture of white-matter tracts in the brain. This method has proved promising in understanding and investigating the neuronal tracts and structural connectivity between the brain regions in primates as well as rodents. The close evolutionary relationship between canines and humans may have spawned a unique bond in regard to social cognition rendering them useful as an animal model in translational research. In this study, we acquired diffusion data from anaesthetized dogs and created a DTI-based atlas for a canine model which could be used to investigate various white matter diseases. We illustrate the application of this atlas by calculating DTI tractography based structural connectivity between the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) regions of the default mode network (DMN) in dogs. White matter connectivity was investigated to provide structural basis for the functional dissociation observed between the anterior and posterior parts of DMN. A comparison of the integrity of long range structural connections (such as in the DMN) between dogs and humans is likely to provide us with new perspectives on the neural basis of the evolution of cognitive functions., Competing Interests: Author N.S. is an employee of Siemens Healthcare, Malvern, PA who is stationed at the MRI Research Center in Auburn University. She contributed by optimizing the sequences used to acquire data in this study. However, note that she had no role in data analysis and its interpretation. These competing interests do not alter the authors’ adherence to common policies on sharing data and materials.

One of the principal challenges in the collection of biological samples from air, water, and soil matrices is that the target agents are not stable enough to be transferred from the collection point to the laboratory of choice without experiencing significant degradation and loss of viability. At present, there is no method to transport biological samples over considerable distances safely, efficiently, and cost-effectively without the use of ice or refrigeration. Current techniques of protection and preservation of biological materials have serious drawbacks. Many known techniques of preservation cause structural damages, so that biological materials lose their structural integrity and viability. We review applications of a novel bacterial preservation process, which is nontoxic and water soluble and allows for the storage of samples without refrigeration. The method is capable of protecting the biological sample from the effects of environment for extended periods of time and then allows for the easy release of these collected biological materials from the protective medium without structural or DNA damage. Strategies for sample collection, preservation, and shipment of bacterial, viral samples are described. The water-soluble polymer is used to immobilize the biological material by replacing the water molecules within the sample with molecules of the biopolymer. The cured polymer results in a solid protective film that is stable to many organic solvents, but quickly removed by the application of the water-based solution. The process of immobilization does not require the use of any additives, accelerators, or plastifiers and does not involve high temperature or radiation to promote polymerization.

In the 1960s Bong Han Kim discovered and characterized a new vascular system. He was able to differentiate it clearly from vascular blood and lymph systems by the use of a variety of methods, which were available to him in the mid-20th century. He gave detailed characterization of the system and created comprehensive diagrams and photographs in his publications. He demonstrated that this system is composed of nodes and vessels, and it was responsible for tissue regeneration. However, he did not disclose in detail his methods. Consequently, his results are relatively obscure from the vantage point of contemporary scientists. The stains that Kim used had been perfected and had been in use for more than 100 years. Therefore, the names of the stains were directed to the explicit protocols for the usage with the particular cells or molecules. Traditionally, it was not normally necessary to describe the method used unless it is significantly deviated from the original method. In this present work, we have been able to disclose staining methods used by Kim.

An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria.

This article presents rapid, sensitive, direct detection of Salmonella Typhimurium on eggshells by using wireless magnetoelastic (ME) biosensors. The biosensor consists of a freestanding, strip-shaped ME resonator as the signal transducer and the E2 phage as the biomolecular recognition element that selectively binds with Salmonella Typhimurium. This ME biosensor is a type of mass-sensitive biosensor that can be wirelessly actuated into mechanical resonance by an externally applied timevarying magnetic field. When the biosensor binds with Salmonella Typhimurium, the mass of the sensor increases, resulting in a decrease in the sensor's resonant frequency. Multiple E2 phage-coated biosensors (measurement sensors) were placed on eggshells spiked with Salmonella Typhimurium of various concentrations (1.6 to 1.6 × 10(7) CFU/cm(2)). Control sensors without phage were also used to compensate for environmental effects and nonspecific binding. After 20 min in a humidity-controlled chamber (95%) to allow binding of the bacteria to the sensors to occur, the resonant frequency of the sensors was wirelessly measured and compared with their initial resonant frequency. The resonant frequency change of the measurement sensors was found to be statistically different from that of the control sensors down to 1.6 × 10(2) CFU/cm(2), the detection limit for this work. In addition, scanning electron microscopy imaging verified that the measured resonant frequency changes were directly related to the number of bound cells on the sensor surface. The total assay time of the presented methodology was approximately 30 min, facilitating rapid detection of Salmonella Typhimurium without any preceding sampling procedures.

Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air-water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of ∼40 mN/m at 20 °C. The surface pressure-area (Π-A) isotherm possessed a shoulder at ∼7 × 10(4)nm(2)/phage particle, attributed to a change in phage orientation at the air-water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Π-A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air-water interface at low surface pressures with an excluded area per phage particle of 4.6 × 10(4)nm(2). At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5 × 10(3)nm(2)/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air-water interface at a constant surface pressure of 18 mN/m by Langmuir-Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Γ) in Langmuir-Blodgett (LB) films measured by SEM was consistent with that calculated independently from Π-A isotherms at the transfer surface pressure of 18 mN/m (Γ=23 phage particles/μm(2)). The 50 nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air-water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Two bacterial cultures were isolated and tested for degradation of shrimp shell waste. According to morphological examination, physiological tests, and applied molecular techniques, isolates were identified as Bacillus cereus and Exiguobacterium acetylicum. Both strains were cultivated separately in flasks with 100 mL of shrimp shell waste broth (3% of washed, dried and ground shrimp shell waste in tap water, pH 7.0) at 37 degrees C. At determined periods of time, deproteinization and demineralization of residuals were measured. Fermentation of 3% shell waste with B. cereus indicated 97.1% deproteinization and 95% demineralization. For E. acetylicum, the level of deproteinization and demineralization was 92.8 and 92%, respectively. Protein content was reduced from 18.7 to 5.3% with B. cereus and to 7.3% with E. acetylicum. No additional supplements were used during the fermentation of shell waste. B. cereus strain showed higher efficacy in decomposition of shell waste and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth. Incubation of bacteria with shell waste during 14 days at 37 degrees C resulted in 78.6% deproteinization and 73% demineralization. High activity of isolated cultures in decomposition of shrimp shell waste suggests broad potential for application of these bacteria in environmentally friendly approaches to chitin extraction from chitin-rich wastes.

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoViva optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29 mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.

The antimycotic agent amphotericin B (AmB) functions by forming complexes with sterols to form ion channels that cause membrane leakage. When AmB and cholesterol mixed at 2:1 ratio were incorporated into phospholipid bilayer membranes formed on the tip of patch pipettes, ion channel current fluctuations with characteristic open and closed states were observed. These channels were also functional in phospholipid membranes formed on nanoporous silicon surfaces. Electrophysiological studies of AmB-cholesterol mixtures that were incorporated into phospholipid membranes formed on the surface of nanoporous (6.5 nm pore diameter) silicon plates revealed large conductance ion channels ( approximately 300 pS) with distinct open and closed states. Currents through the AmB-cholesterol channels on nanoporous silicon surfaces can be driven by voltage applied via conventional electrical circuits or by photovoltaic electrical potential entirely generated when the nanoporous silicon surface is illuminated with a narrow laser beam. Electrical recordings made during laser illumination of AmB-cholesterol containing membrane-coated nanoporous silicon surfaces revealed very large conductance ion channels with distinct open and closed states. Our findings indicate that nanoporous silicon surfaces can serve as mediums for ion-channel-based biosensors. The photovoltaic properties of nanoporous silicon surfaces show great promise for making such biosensors addressable via optical technologies.

The antimycotic activity of amphotericin B (AmB) depends on its ability to make complexes sterols to form ion channels that cause membrane leakage. To study this phenomenon, surface pressure (pi) as a function of surface area (A) and pi-A hysteresis were measured in monolayers of AmB-cholesterol mixtures on the water-air interface. The most stable monolayers were produced from molecules of AmB and cholesterol with 2:1 stoichiometry. At this ratio, AmB and cholesterol interact to form ion channels in lipid bilayers with millisecond dwell times and conductances of 4-400 pS. The AmB-cholesterol complexes assemble in three, four, etc., subunit aggregates to form ion channels of diverse and large-conductances. Their I-V characteristics were linear over a range of +/-200 mV. The channel currents were inhibited by the addition of tetraethylammonium (TEA), potassium channel blocker, to the cis-side of the membrane. Likewise, AmB-cholesterol complexes reconstituted in membrane-coated nanoporous silicon dioxide surfaces showed single channel behavior with large amplitudes at various voltages. Large-conductance ion channels show great promise for use in biosensors on solid supports.

Biosensors based on landscape phages immobilized by physical adsorption on the surface of a quartz crystal microbalance was used for detection of beta-galactosidase from Escherichia coli. The sensor had a detection limit of a few nanomoles and a response time of a approximately 100 s over the range of 0.003-210 nM. The binding dose-response curve had a typical sigmoid shape and the signal was saturated at the beta-galactosidase concentration of about 200 nM. A marked selectivity for beta-galactosidase over BSA was observed in mixed solutions even when the concentration of BSA exceeded the concentration of beta-galactosidase by a factor of approximately 2000. The apparent value of the dissociation constant (K(d)) of the interaction of free phage with beta-galactosidase (9.1+/-0.9 pM) was smaller compared with the one calculated for the bound phage (1.7+/-0.5 nM). The binding was specific with three binding sites needed to bind a single molecule of beta-galactosidase. The K(d) obtained from the enzyme-linked immunosorbent assay (ELISA) for the phage and the monoclonal anti-beta-galactosidase antibodies were 21+/-2 and 26+/-2 nM, respectively. Although the method of physical adsorption is simpler and more economical in comparison with Langmuir-Blodgett and molecular assembling methods the performances of the sensors made by these technologies compare well. This work provides evidence that phage can be used as a recognition element in biosensors using physical adsorption method for immobilization of phage on the sensor surface.

Surface-pressure (Pi) and surface-area isotherms as a function of surface area were measured for monolayers of amphotericin B (AmB) and cholesterol mixtures at the air/water interface at 10, 20, and 30 degrees C. When chloroform/methanol was used as a spreading solvent, the Pi-A isotherms of the mixed monolayers exhibited characteristic transitions from the gas to liquid-expanded, then liquid-condensed, and finally the solid state. The expanding effect in monolayers was accompanied by a large Pi-A hysteresis and a positive excess of free energy of mixing at high Pi. At low Pi, a condensing effect was observed with the most significant deviation from ideality occurring at a mole fraction of AmB (XAmB) of 0.67. Free energy calculations revealed a condensing effect at low Pi and an expanding effect at high Pi except at 30 degrees C, where a condensing effect was observed for XAmB around 0.33. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 degrees C exhibited Pi-A isotherms which obey van der Waals equation of state, with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from mixtures of AmB and cholesterol with a 2:1 stoichiometry.

Monolayers of the antifungal antibiotic amphotericin B undergo the liquid expanded/liquid condensed state transition if spread from chloroform/methanol solvent. The transition disappeared after a long spreading time. The presence of the transition may be due to the retention of solvent and/or the presence of metastable aggregates of amphotericin B.

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.

The odorant-induced accumulation of cAMP can be inhibited by antibodies directed against G alpha s/olf. In contrast, antibodies raised against G alpha i-subunits caused a strong enhancement of the odorant-induced cAMP accumulation. Western blotting and immunoelectron microscopy revealed the presence of both G alpha s/olf- and G alpha i-subunits in rat cilia preparations. The existence of both stimulatory and inhibitory odorant-induced regulation of adenylyl cyclase activity in olfactory cilia may indicate that an initial integration of different odorant stimuli begins at the level of primary reactions in the same effector enzyme.

Chemosensitive single-channel fluctuations were observed to be induced in essentially solvent-free lipid bimolecular membranes by the addition of sonicated homogenates of rat olfactory epithelium. The chemosensitive channels were not observed when respiratory epithelium homogenates were used instead. Ionic selectivity is consistent with potassium ions as the charge carrier. These channels may be associated with the initial events of chemoreception in the rat olfactory epithelium.