A nanoelectronics-blood-based diagnostic biomarker for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

There is not currently a well-established, if any, biological test to diagnose myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The molecular aberrations observed in numerous studies of ME/CFS blood cells offer the opportunity to develop a diagnostic assay from blood samples. Here we developed a nanoelectronics assay designed as an ultrasensitive assay capable of directly measuring biomolecular interactions in real time, at low cost, and in a multiplex format. To pursue the goal of developing a reliable biomarker for ME/CFS and to demonstrate the utility of our platform for point-of-care diagnostics, we validated the array by testing patients with moderate to severe ME/CFS patients and healthy controls. The ME/CFS samples' response to the hyperosmotic stressor observed as a unique characteristic of the impedance pattern and dramatically different from the response observed among the control samples. We believe the observed robust impedance modulation difference of the samples in response to hyperosmotic stress can potentially provide us with a unique indicator of ME/CFS. Moreover, using supervised machine learning algorithms, we developed a classifier for ME/CFS patients capable of identifying new patients, required for a robust diagnostic tool.

An electrochemical platform for localized pH control on demand.

Solution pH is a powerful tool for regulating many kinds of chemical activity, but is generally treated as a static property defined by a pre-selected buffer. Introducing dynamic control of pH in space, time, and magnitude can enable richer and more efficient chemistries, but is not feasible with traditional methods of titration or buffer exchange. Recent reports have featured electrochemical strategies for modifying bulk pH in constrained volumes, but only demonstrate switching between two preset values and omit spatial control entirely. Here, we use a combination of solution-borne quinones and galvanostatic excitation to enable quantitative control of pH environments that are highly localized to an electrode surface. We demonstrate highly reproducible acidification and alkalinization with up to 0.1 pH s(-1) (±0.002 pH s(-1)) rate of change across the dynamic range of our pH sensor (pH 4.5 to 7.5) in buffered solutions. Using dynamic current control, we generate and sustain 3 distinct pH microenvironments simultaneously to within ±0.04 pH for 13 minutes in a single solution, and we leverage these microenvironments to demonstrate spatially-resolved, pH-driven control of enzymatic activity. In addition to straightforward applications of spatio-temporal pH control (e.g. efficiently studying pH-dependencies of chemical interactions), the technique opens completely new avenues for implementing complex systems through dynamic control of enzyme activation, protein binding affinity, chemical reactivity, chemical release, molecular self-assembly, and many more pH-controlled processes.

A novel low energy electron microscope for DNA sequencing and surface analysis.

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts. The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence.

Resting metabolic rate and heat increment of feeding in juvenile South American fur seals (Arctocephalus australis).

Bio-energetic models used to characterize an animal's energy budget require the accurate estimate of different variables such as the resting metabolic rate (RMR) and the heat increment of feeding (HIF). In this study, we estimated the in air RMR of wild juvenile South American fur seals (SAFS; Arctocephalus australis) temporarily held in captivity by measuring oxygen consumption while at rest in a postabsorptive condition. HIF, which is an increase in metabolic rate associated with digestion, assimilation and nutrient interconversion, was estimated as the difference in resting metabolic rate between the postabsorptive condition and the first 3.5h postprandial. As data were hierarchically structured, linear mixed effect models were used to compare RMR measures under both physiological conditions. Results indicated a significant increase (61%) for the postprandial RMR compared to the postabsorptive condition, estimated at 17.93±1.84 and 11.15±1.91mL O2 min(-1)kg(-1), respectively. These values constitute the first estimation of RMR and HIF in this species, and should be considered in the energy budgets for juvenile SAFS foraging at-sea.

Depletion of cells and abundant proteins from biological samples by enhanced dielectrophoresis.

Platforms that are sensitive and specific enough to assay low-abundance protein biomarkers, in a high throughput multiplex format, within a complex biological fluid specimen, are necessary to enable protein biomarker based diagnostics for diseases such as cancer. The signal from an assay for a low-abundance protein biomarker in a biological fluid sample like blood is typically buried in a background that arises from the presence of blood cells and from high-abundance proteins that make up 90% of the assayed protein mass. We present an automated on-chip platform for the depletion of cells and highly abundant serum proteins in blood. Our platform consists of two components, the first of which is a microfluidic mixer that mixes beads containing antibodies against the highly abundant proteins in the whole blood. This complex mixture (consisting of beads, cells, and serum proteins) is then injected into the second component of our microfluidic platform, which comprises a filter trench to capture all the cells and the beads. The size-based trapping of the cells and beads into the filter trench is significantly enhanced by leveraging additional negative dielectrophoretic forces to push the micron sized particles (cells and beads which have captured the highly abundant proteins) down into the trench, allowing the serum proteins of lower abundance to flow through. In general, dielectrophoresis using bare electrodes is incapable of producing forces beyond the low piconewton range that tend to be insufficient for separation applications. However, by using electrodes passivated with atomic layer deposition, we demonstrate the application of enhanced negative DEP electrodes together with size-based flltration induced by the filter trench, to deplete 100% of the micron sized particles in the mixture.

A microfluidic platform for electrical detection of DNA hybridization.

Current methods used for detection of DNA hybridization involve the use of DNA microarrays which require overnight incubation times along with bulky and expensive fluorescent scanners. Here, we demonstrate electrical detection of DNA hybridization in an oligonucleotide functionalized microfluidic channel. We use microchannels functionalized with DNA probes integrated with electrodes for measuring conductance across the channel. As beads conjugated with the target DNA passing through the channel are captured on the surface, we are able to electrically detect changes in resistance due to bead capture. Our assay can be completed in less than an hour using less than a microliter of reagent, and has the potential for extensive multiplexing. Such a device can be useful as a handheld platform in a clinical setting where one would need to rapidly genotype a small number of genes rapidly.

Microfluidic force spectroscopy for characterization of biomolecular interactions with piconewton resolution.

In this paper we present a scalable method based on the use of microfluidics and shear force spectroscopy which can be used for determining the affinity between molecules. Our method involves the use of functionalization of the surface of microfluidic channels with ligand molecules, and the surface of microspheres with receptor molecules. Bound beads are detached from the surface of the microchannels using pressure driven flow. The drag force required to detach the beads is used to determine the affinity of the bond holding the two molecules together. The minimum force we are able to detect is 5 pN. We have used this method to determine the binding force between protein-protein interactions and DNA base-pair interactions. We also have shown the ability of this technique to distinguish between strong and weak protein-protein interactions. Using this approach, it may be possible to multiplex an array of these functionalized channels onto a chip and probe the interactions between large varieties of biomolecules.

The effects of water temperature on the energetic costs of juvenile and adult California sea lions (Zalophus californianus): the importance of skeletal muscle thermogenesis for thermal balance.

As highly mobile marine predators, many pinniped species routinely encounter a wide range of water temperatures during foraging and in association with seasonal, geographical and climatic changes. To determine how such variation in environmental temperature may impact energetic costs in otariids, we determined the thermal neutral zone of adult and juvenile California sea lions (Zalophus californianus) by measuring resting metabolic rate using open-flow respirometry. Five adult female (body mass range =82.2-107.2 kg) and four juvenile (body mass=26.2-36.5 kg) sea lions were examined over experimental water temperatures ranging from 0 to 20 degrees C (adults) or 5 to 20 degrees C (juveniles). The metabolic rate of adult sea lions averaged 6.4+/-0.64 ml O(2) kg(-1) min(-1) when resting within the thermal neutral zone. The lower critical temperature of adults was 6.4+/-2.2 degrees C, approximately 4 degrees C lower than sea surface temperatures routinely encountered off coastal California. In comparison, juvenile sea lions did not demonstrate thermal neutrality within the range of water temperatures examined. Resting metabolic rate of the younger animals, 6.3+/-0.53 ml O(2) kg(-1) min(-1), increased as water temperature approached 12 degrees C, and suggested a potential thermal limitation in the wild. To determine whether muscle thermogenesis during activity could mitigate this limitation, we measured the active metabolic rate of juveniles swimming at water temperature (T(water))=5, 12 and 20 degrees C. No significant difference (F=0.377, P=0.583) in swimming metabolic rate was found among water temperatures, suggesting that thermal disadvantages due to small body size in juvenile sea lions may be circumvented by recycling endogenous heat during locomotor activity.

Contact versus feeding intoxication by fipronil in Reticulitermes termites (Isoptera: Rhinotermitidae): laboratory evaluation of toxicity, uptake, clearance, and transfer among individuals.

The susceptibility to fipronil of U.S. and French populations of Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) was evaluated in two types of laboratory bioassays: contact intoxication with 0.01-1 ppm treated sand and feeding intoxication with 0.1-10 ppm treated filter paper. Contact intoxication with 0.01 ppm fipronil caused 60% mortality after 55 and 64 h in the French and U.S. populations, respectively, whereas in the 5 ppm feeding assay 70 and 60% mortality was observed after 7 d in the French and U.S. populations, respectively. We evaluated the uptake, clearance, and transfer of fipronil among workers of French R. flavipes using [14C] fipronil in contact (0.01 ppm) and feeding (3 ppm) bioassays. Fipronil amounts were measured on their cuticle and in their bodies. Maximal uptake was observed in the contact assay. A significant uptake of fipronil occurred in the feeding assay. Transfer from exposed donors to unexposed recipients occurred within 24 h. Frequent horizontal transfer resulted in a significant uptake in recipients, particularly when donor fipronil acquisition was by feeding. Donors transferred approximately 46% of the toxicant to recipients. Social behaviors such as contact and grooming, together with internalization of the biocide, may be components of the horizontal transfer process and contribute to the efficacy of fipronil in the field.

Type III ossiculoplasty with mastoid cortical bone--"in situ" shaping of the ossicle.

The authors describe their technique to harvest a cortical bone ossicle. It consists of sculpting and shaping the ossicle while it is still attached to cortical bone. The technique is simple and safe.

Electrical Detection of Proteins and DNA using Bioactivated Microfluidic Channels: Theoretical and Experimental Considerations.

In order to detect diseases like cancer at an early stage while it still may be curable, it's necessary to develop a diagnostic technique which can rapidly and inexpensively detect protein and nucleic acid biomarkers, without making any sacrifice in the sensitivity. We have developed a technique, based on the use of bioactivated microfluidic channels integrated with electrodes for electrical sensing, which can be used to detect protein biomarkers, target cells, and DNA hybridization. In this paper, we discuss the theoretical detection limits of this kind of sensor, and also discuss various experimental considerations in the electrical characterization of our device. In particular, we discuss the temperature dependence, the impedance drift, the noise sources, and various methods for optimizing the signal to noise ratio.

The ontogeny of aerobic and diving capacity in the skeletal muscles of Weddell seals.

Our objective was to determine the ontogenetic changes in the skeletal muscles of Weddell seals that transform a non-diving pup into an elite diving adult. Muscle biopsies were collected from pups, juveniles and adults and analyzed for changes in fiber type, mitochondrial density, myoglobin concentrations and aerobic, lipolytic and anaerobic enzyme activities. The fiber type results demonstrated a decrease in slow-twitch oxidative (Type I) fibers and a significant increase in fast-twitch oxidative (Type IIA) fibers as the animals mature. In addition, the volume density of mitochondria and the activity of lipolytic enzymes significantly decreased as the seals matured. To our knowledge, this is the first quantitative account describing a decrease in aerobic fibers shifting towards an increase in fast-twitch oxidative fibers with a significant decrease in mitochondrial density as animals mature. These differences in the muscle physiology of Weddell seals are potentially due to their three very distinct stages of life history: non-diving pup, novice diving juvenile, and elite deep diving adult. During the first few weeks of life, pups are a non-diving terrestrial mammal that must rely on lanugo (natal fur) for thermoregulation in the harsh conditions of Antarctica. The increased aerobic capacity of pups, associated with increased mitochondrial volumes, acts to provide additional thermogenesis. As these future elite divers mature, their skeletal muscles transform to a more sedentary state in order to maintain the low levels of aerobic metabolism associated with long-duration diving.

The effect of myoglobin concentration on aerobic dive limit in a Weddell seal.

One physiological adaptation for prolonged dive duration in marine mammals is an elevated myoglobin (Mb) concentration in skeletal muscle. To determine the influence of Mb concentration on the aerobic dive limit (ADL), we modified a previously published model that simulated aerobic dives in a Weddell seal (Leptonychotes weddellii) and ran it for four Mb concentrations: 5, 27, 54 and 108 g Mb kg(-1) muscle representing 7%, 50%, 100% and 200%, respectively, of the normal Mb concentration in Weddell seal skeletal muscle. The model was run at increasing levels of muscular exertion and under postabsorptive and postprandial conditions to determine their effect on ADL. For each set of conditions, the model was also run at different levels of cardiac output (i.e. the dive response was varied) to determine the level of convective oxygen transport that optimized the ADL. In a postabsorptive state at a routine level of muscular exertion for a diving Weddell seal, a decrease in Mb concentration to 7% of normal caused a 39% decrease in the ADL (18 min to 11 min), while doubling the Mb concentration increased the ADL by 30% (18 min to 24 min). Under postprandial conditions at a routine level of muscular exertion, doubling the Mb concentration did not increase the ADL (12 min). The convective oxygen transport needed to meet the metabolic demands (Heat Increment of Feeding, HIF) of the splanchnic organs during digestion and assimilation required a cardiac output that was not optimal for the efficient use of muscle oxygen stores. This resulted in an over perfusion of the muscles and incomplete use of myoglobin-bound oxygen. As a result, the postprandial ADL was limited by the amount of oxygen stored in the blood, and increasing the Mb concentration had no effect on the ADL. We hypothesize that myoglobin concentration is optimized for the type and duration of dives routinely made by Weddell seals, and that a further increase may not increase the ADL for most free-ranging dives.

Metabolic indicators in the skeletal muscles of harbor seals (Phoca vitulina).

The goal of this study was to determine the distribution of citrate synthase (CS), beta-hydroxyacyl coenzyme A dehydrogenase (HOAD), and lactate dehydrogenase (LDH) activities and myoglobin (Mb) concentration in the locomotor muscles (epaxial muscles) and heart of harbor seals. The entire epaxial musculature, which produces most of the power for submerged swimming, was removed and weighed, and three transverse sections (cranial, middle, and caudal) were taken along the muscle bundle. Multiple samples were taken along points on a circular grid using a 6-mm biopsy. A single sample was taken from the left ventricle of the heart. Muscle groups of similar function were taken from three dogs as a control. Mean values were calculated for four roughly equal quadrants in each transverse section of the epaxial muscles. There were no significant differences among the quadrants within any of the transverse sections for the three enzymes or Mb. However, there were significant differences in the mean enzyme activities and Mb concentrations along the length of the muscle. The middle and caudal sections had significantly higher mean levels of CS, LDH, and Mb than the cranial section, which may be correlated with power production during swimming. The enzyme ratios CS/HOAD and LDH/CS exhibited no variation within transverse sections or along the length of the epaxial muscles. Relative to the dog, the epaxial muscles and heart of the harbor seal had higher HOAD levels and lower CS/HOAD, which, taken together, indicate an increased capacity for aerobic lipid metabolism during diving.

General practitioners' educational needs in intellectual disability health.

BACKGROUND: The community general practitioner (GP) has a central role in the provision of primary health care to people with intellectual disability (ID) as an indirect result of deinstitutionalization in Australia. This population, however, continues to experience poor health care compared to the general population. The current paper describes results from a questionnaire that aimed to identify the perceptions of practising GPs on the standards of health care for people with ID, the adequacy of prior training, and their interest in further education in relation to nine health care areas. METHOD: A questionnaire was posted to a selective sample of 1272 practising GPs in Victoria selected from a database from the Centre for Developmental Disability Health Victoria and the Victorian Medical Directory of GPs registered with the Australian Medical Association. Data were available for 252 respondents with a response rate of 28.5%. RESULTS: The health areas in which many GPs reported to be inadequately trained were the same as those areas that were perceived as being of a poor standard. These areas were behavioural or psychiatric conditions, human relations and sexuality issues, complex medical problems, and preventative and primary health care. Ninety four per cent of respondents were interested in further education in at least one of the nine health care areas, with the most frequently nominated areas being behavioural or psychiatric conditions, syndrome-specific medical problems, human relations and sexuality issues and collaboration with government services. General practitioners did not nominate complex medical problems or preventative and primary health care for further education as frequently as they identified care in these areas to be substandard and their prior training inadequate. CONCLUSIONS: The findings from the current research are discussed in relation to the implications for development of educational programmes based on learning needs identified by the GP. The most frequently nominated health care areas in all three questions were behavioural or psychiatric conditions and human relations and sexuality issues. Reasons for incongruence between the frequency of responses for complex medical problems and preventative and primary health care are explored.

Region-specific changes in immediate early gene expression in response to sleep deprivation and recovery sleep in the mouse brain.

Previous studies have documented changes in expression of the immediate early gene (IEG) c-fos and Fos protein in the brain between sleep and wakefulness. Such expression differences implicate changes in transcriptional regulation across behavioral states and suggest that other transcription factors may also be affected. In the current study, we examined the expression of seven fos/jun family member mRNAs (c-fos, fosB, fos related antigen (fra)1, fra-2, junB, c-jun, and junD) and three other IEG mRNAs (egr-1, egr-3, and nur77) in mouse brain following short-term (6 h) sleep deprivation (SD) and 4 h recovery sleep (RS) after SD. Gene expression was quantified in seven brain regions by real-time reverse transcription-polymerase chain reaction (RT-PCR). Multivariate analysis of variance revealed statistically significant variation in cerebral cortex, basal forebrain, thalamus and cerebellum. Levels of c-fos and fosB mRNA were elevated during SD in all four of these brain regions. In the cerebral cortex, junB mRNA was also elevated during SD whereas, in the basal forebrain, fra-1 and fra-2 mRNA levels increased in this condition. During RS, the only IEG mRNA to undergo significant increase was fra-2 in the cortex. C-jun and junD mRNAs were invariant across experimental conditions. These results indicate that the expression of fos/jun family members is diverse during SD. Among other IEGs, nur77 mRNA expression across conditions was similar to c-fos and fosB, egr-1 mRNA was elevated during SD in the cortex and basal forebrain, and egr-3 mRNA was elevated in the cortex during both SD and RS. The similarity of fosB and nur77 expression to c-fos expression indicates that these genes might also be useful markers of functional activity. Along with our previous results, the increased levels of fra-2 and egr-3 mRNAs during RS reported here suggest that increased mRNA expression during sleep is rare and may be anatomically restricted.

Differential increase in the expression of heat shock protein family members during sleep deprivation and during sleep.

Although sleep is thought to be restorative from prior wakeful activities, it is not clear what is being restored. To determine whether the synthesis of macromolecules is increased in the cerebral cortex during sleep, we subjected C57BL/6 mice to 6 hours of sleep deprivation and then screened the expression of 1176 genes of known function by using cDNA arrays. The expression of the heat shock proteins (HSP), endoplasmic reticulum protein (ERp72) and glucose-regulated protein (GRp78), was among the genes whose expression was significantly elevated in the cortex during sleep deprivation, whereas GRp78 and GRp94 mRNAs were elevated in the cortex during recovery sleep after sleep deprivation, as confirmed by conventional and quantitative real-time polymerase chain reaction and/or Northern analyses. A systematic evaluation of the expression of six heat shock protein family members (ERP72, GRp78, GRp94, HSP27, HSP70-1, and HSP84) in seven brain regions revealed increased mRNA levels in cortex, basal forebrain, hypothalamus, cerebellum and medulla during sleep deprivation, whereas increased mRNA levels during recovery sleep were limited to the cortex and medulla. Immunohistochemical studies identified increased numbers of GRp78-, GRp94-, and ERp72-immunoreactive cells in the dorsal and lateral cortex during sleep deprivation but, during recovery sleep, elevated numbers of these cells were found only in the lateral cortex. In the medulla, increased numbers of GRp94-immunoreactive cells were observed in nucleus tractus solitarius, dorsal motor nucleus of the vagus and the rostroventrolateral medulla during recovery sleep. The widespread increase of heat shock protein family mRNAs in brain during sleep deprivation may be a neuroprotective response to prolonged wakefulness. In contrast, the relatively limited heat shock protein family mRNA expression during recovery sleep may be related to the role of heat shock proteins in protein biogenesis and thus to the restorative function of sleep.

Aerobic capacities in the skeletal muscles of Weddell seals: key to longer dive durations?

In contrast to terrestrial animals that function under hypoxic conditions but display the typical exercise response of increasing ventilation and cardiac output, marine mammals exercise under a different form of hypoxic stress. They function for the duration of a dive under progressive asphyxia, which is the combination of increasing hypoxia, hypercapnia and acidosis. Our previous studies on short-duration, shallow divers found marked adaptations in their skeletal muscles, which culminated in enhanced aerobic capacities that are similar to those of athletic terrestrial mammals. The purpose of the present study was to assess the aerobic capacity of skeletal muscles from long-duration divers. Swimming and non-swimming muscles were collected from adult Weddell seals, Leptonychotes weddelli, and processed for morphometric analysis, enzymology, myoglobin concentrations and fiber-type distribution. The results showed that the skeletal muscles of Weddell seals do not have enhanced aerobic capacities compared with those of terrestrial mammals but are adapted to maintain low levels of an aerobic lipid-based metabolism, especially under the hypoxic conditions associated with diving. The lower aerobic capacity of Weddell seal muscle as compared with that of shorter-duration divers appears to reflect their energy-conserving modes of locomotion, which enable longer and deeper dives.

A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity.

The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes. Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array. We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes). We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage. We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted. Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.

Influence of Voltage-sensitive Ca(++) channel drugs on bupivacaine infiltration anesthesia in mice.

BACKGROUND: Local anesthesia has been traditionally associated with blockade of voltage-sensitive sodium (Na(+)) channels. Yet in vitro evidence indicates that local anesthetic mechanisms are more complex than previously understood. For example, local anesthetics bind and allosterically modify 1,4-dihydropyridine-sensitive Ca(++) channels and can reduce Ca(++) influx in tissues. The current study examines the influence of voltage-sensitive Ca(++) channels in bupivacaine infiltration anesthesia. METHODS: Baseline tail-flick latencies to radiant heat nociception were obtained before subcutaneous infiltration of bupivacaine and Ca(++)-modulating drugs in the tails of mice. No musculature is contained in the tail that could result in motor block. The magnitude of infiltration anesthesia over time, as well as the potency of bupivacaine alone or in the presence of Ca(++)-modulating drug, was assessed by obtaining test latencies. RESULTS: The 1,4-dihydropyridine L-type Ca(++) channel agonist S(-)-BayK-8644 reduced the duration of action and potency of bupivacaine anesthesia. In opposite fashion, nifedipine and nicardipine increased the effects of bupivacaine. Neither nifedipine nor nicardipine alone elicited anesthesia. Alternatively, the phenylalkylamine L-type blocker verapamil elicited concentration-dependent anesthesia. Other Ca(++) channel subtype blockers were investigated as well. The N-, T-, P-, and Q-type channel blockers, omega-conotoxin GVIA, flunarizine, omega-agatoxin IVA, and omega-conotoxin MVIIC, respectively, were unable to modify bupivacaine anesthesia. CONCLUSIONS: These results indicate that heat nociception stimulates Ca(++) influx through L-type channels on nociceptors in skin. Although other voltage-sensitive Ca(++) channels may be located on skin nociceptors, only the L-type channel drugs affected bupivacaine in the radiant heat test.