Estimating Population Abundance of Burying Beetles Using Photo-Identification and Mark-Recapture Methods.

Successful conservation and management of protected wildlife populations require reliable population abundance data. Traditional capture-mark-recapture methods can be costly, time-consuming, and invasive. Photographic mark-recapture (PMR) is a cost-effective, minimally invasive way to study population dynamics in species with distinct markings or color patterns. We tested the feasibility and the application of PMR using the software Hotspotter to identify Nicrophorus spp. from digital images of naturally occurring spot patterns on their elytra. We conducted a laboratory study evaluating the identification success of Hotspotter on Nicrophorus americanus (Olivier, 1790) and Nicrophorus orbicollis (Say, 1825) before implementation of a mark-recapture study in situ. We compared the performance of Hotspotter using both 'high-quality' and 'low-quality' photographs. For high-quality photographs, Hotspotter had a false rejection rate of 2.7-3.0% for laboratory-reared individuals and 3.9% for wild-caught individuals. For low-quality photographs, the false rejection rate was much higher, 48.8-53.3% for laboratory-reared individuals and 28.3% for wild-caught individuals. We subsequently analyzed encounter histories of wild-caught individuals with closed population models in Program MARK to estimate population abundance. In our study, we demonstrated the utility of using PMR in estimating population abundance for Nicrophorus spp. based on elytral spot patterns.

Behavioral constraints on local adaptation and counter-gradient variation: Implications for climate change.

Resource allocation to growth, reproduction, and body maintenance varies within species along latitudinal gradients. Two hypotheses explaining this variation are local adaptation and counter-gradient variation. The local adaptation hypothesis proposes that populations are adapted to local environmental conditions and are therefore less adapted to environmental conditions at other locations. The counter-gradient variation hypothesis proposes that one population out performs others across an environmental gradient because its source location has greater selective pressure than other locations. Our study had two goals. First, we tested the local adaptation and counter-gradient variation hypotheses by measuring effects of environmental temperature on phenotypic expression of reproductive traits in the burying beetle, Nicrophorus orbicollis Say, from three populations along a latitudinal gradient in a common garden experimental design. Second, we compared patterns of variation to evaluate whether traits covary or whether local adaptation of traits precludes adaptive responses by others. Across a latitudinal range, N. orbicollis exhibits variation in initiating reproduction and brood sizes. Consistent with local adaptation: (a) beetles were less likely to initiate breeding at extreme temperatures, especially when that temperature represents their source range; (b) once beetles initiate reproduction, source populations produce relatively larger broods at temperatures consistent with their local environment. Consistent with counter-gradient variation, lower latitude populations were more successful at producing offspring at lower temperatures. We found no evidence for adaptive variation in other adult or offspring performance traits. This suite of traits does not appear to coevolve along the latitudinal gradient. Rather, response to selection to breed within a narrow temperature range may preclude selection on other traits. Our study highlights that N. orbicollis uses temperature as an environmental cue to determine whether to initiate reproduction, providing insight into how behavior is modified to avoid costly reproductive attempts. Furthermore, our results suggest a temperature constraint that shapes reproductive behavior.

Comparison of the performance of three variations of the Carbapenem Inactivation Method (CIM, modified CIM [mCIM] and in-house method (iCIM)) for the detection of carbapenemase-producing Enterobacterales and non-fermenters.

OBJECTIVE: The objective of this study was to compare the performance of an in-house CIM (iCIM) modification with the CIM and mCIM for the detection of carbapenemase production in 149 well characterised isolates (70 carbapenemase producers and 79 non-carbapenemase producers). METHODS: Isolates were tested using the CIM, mCIM and iCIM procedures. The gold standard was genotypic characterisation by PCR. RESULTS: For Acinetobacter baumannii, the sensitivity was low (10%) for the mCIM, 70% for the CIM but was 100% for the iCIM, with a specificity of 100% for all three. For Enterobacterales, the sensitivity of all three tests was 100% for Ambler class A and B ß-lactamases, while the iCIM also had a sensitivity of 100% for class D ß lactamases. The sensitivity in Enterobacterales was highest for the iCIM at 100% (CIM 98.2%, mCIM 96.2%). The specificity was 100% for the mCIM and 98% for the CIM and iCIM. For Pseudomonas aeruginosa, the sensitivity of the CIM (100%) was higher than the iCIM (85.7%) and the mCIM (71.4%). iCIM exhibited excellent sensitivity (100%) and specificity (98%) for carbapenemase detection in Enterobacterales and was able to detect two OXA-232 producers that the mCIM did not detect and an OXA-181 producer that the CIM did not detect. CONCLUSIONS: In conclusion, iCIM performed better than the CIM and mCIM for carbapenemase detection in A. baumannii and Enterobacterales, however the CIM achieved the highest sensitivity for carbapenemase detection in P. aeruginosa suggesting that different CIM variations should be utilised depending on the organism type.

Isotopic discrimination between carrion and elytra clippings of lab-reared American burying beetles (Nicrophorus americanus): Implications for conservation and evaluation of feeding relationships in the wild.

RATIONALE: Differences in stable isotope composition between an animal and its diet are quantified by experimentally derived diet-tissue discrimination factors. Appropriate discrimination factors between consumers and prey are essential for interpreting stable isotope patterns in ecological studies. While available for many taxa, these values are rarely estimated for organisms within the carrion food web. METHODS: We used a controlled-diet stable isotope feeding trial to quantify isotopic diet-tissue discrimination factors of carbon (δ13 C values) and nitrogen (δ15 N values) from laboratory-reared Nicrophorus americanus raised on carrion. We used exoskeleton samples of beetle elytra (wing covers) to determine diet-tissue discrimination factors using a continuous flow isotope ratio mass spectrometer equipped with an elemental analyzer. We also measured the isotopic compositions of five species of co-occurring, wild-caught burying beetles and evaluated feeding relationships. RESULTS: We found differences in stable carbon discrimination between carrion sources (mammalian and avian) and lab-reared beetles, but no difference in stable nitrogen discrimination. Values for δ13 C did not differ among wild-caught burying beetle species, but values for δ15 N were significantly different for the three species with overlapping breeding seasons. Furthermore, wild-caught burying beetles within our study area do not appear to use avian carrion resources to rear their young. CONCLUSIONS: This study informs future interpretation of stable isotope data for insects within the carrion food web. In addition, these results provide insight into carrion resources used by co-occurring burying beetle species in situ. We also demonstrated that independent of adult food type, the larval food source has a significant impact on the isotopic signatures of adult beetles, which can be estimated using a minimally invasive elytra clipping.

Examining transmission of gut bacteria to preserved carcass via anal secretions in Nicrophorus defodiens.

Direct transmission of bacteria to subsequent generations highlights the beneficial nature of host-bacteria relationships. In insects, this process is often mediated by the production of microbe-containing secretions. The objective of this study was to determine if the burying beetle, Nicrophorus defodiens, utilizes anal secretions to transmit adult digestive tract bacteria onto a small vertebrate carcass; thus creating the potential to aid in carcass preservation or pass digestive tract bacteria to their larval offspring. Using high-throughput Illumina sequencing of the 16S rRNA gene, we characterized bacterial communities of adult beetle digestive tracts, their anal secretions, and prepared mouse carcasses. We also examined unprepared carcass bacterial communities as a means to interpret community shifts that take place during carcass preservation. We found a vast reduction in diversity on prepared carcasses after anal secretion application. Overall, there was little similarity in bacterial communities among adult digestive tracts, anal secretions, and prepared carcasses, suggesting bacterial communities found in adult digestive tracts do not successfully colonize and achieve dominance on prepared carcasses by way of beetle anal secretions. We concluded that N. defodiens does not transmit their digestive tract bacterial communities to prepared carcasses in a wholesale manner, but may transmit key microbes, including core microbiome members, to preserved carcasses that may ultimately act to sustain larvae and serve as inocula for larval digestive tracts.

Why does it take two to tango? Lifetime fitness consequences of parental care in a burying beetle.

In species that require parental care, each parent can either care for their offspring or leave them in the care of the other parent. For each parent this creates three possible parental care strategies: biparental care, uniparental (male or female) care, and uniparental desertion by either the male or female. The burying beetle, Nicrophorus orbicollis, typically exhibits biparental care of offspring, and thus provides a unique system that allows us to compare the fitness benefits of these parental care strategies in an unconfounded way. In this study, we assess the lifetime fitness of biparental care, uniparental care, and uniparental desertion strategies in both male and female N. orbicollis. Specifically, we tested for increased fitness of the biparental care strategy compared to uniparental care strategies. Second, we test for equality of fitness between uniparental care and uniparental desertion strategies. Surprisingly, biparental care yields lower lifetime fitness for both parents compared to the other two strategies. Also, uniparental care and uniparental desertion strategies yielded equal fitness. The evolution of biparental care in this system is not consistent with the expectation of a mutual fitness benefit. We discuss other potential explanations for the evolution of biparental care in this system.

A Comparison of Five Brief Screening Tools for HIV-Associated Neurocognitive Disorders in the USA and South Africa.

Screening for HIV-associated neurocognitive disorders (HAND) is important to improve clinical outcomes. We compared the diagnostic sensitivity and specificity of the mini-mental state examination, International HIV dementia scale (IHDS), Montreal cognitive assessment, Simioni symptom questionnaire and cognitive assessment tool-rapid version (CAT-rapid) to a gold standard neuropsychological battery. Antiretroviral-experienced participants from Cape Town, South Africa, and Baltimore, USA, were recruited. The sensitivity and specificity of the five tools, as well as those of the combined IHDS and CAT-rapid, were established using 2 × 2 contingency tables and ROC analysis. More than a third (65165) had symptomatic HAND. In detecting HIV-D, the CAT-Rapid had good sensitivity (94 %) and weak specificity (52 %) (cut-point ≤10), while the IHDS showed fair sensitivity (68 %) and good specificity (86 %) (cut-point ≤10). The combined IHDS and CAT-rapid showed excellent sensitivity and specificity for HIV-D at a cut-off score of ≤16 (out of 20; 89 and 82 %). No tool was adequate in screening for any HAND. The combination IHDS and CAT-rapid tool appears to be a good screener for HIV-D but is only fairly sensitive and poorly specific in screening for any HAND. Screening for milder forms of HAND continues to be a clinical challenge.

Differences in Patterns of Reproductive Allocation between the Sexes in Nicrophorus orbicollis.

Organisms are selected to maximize lifetime reproductive success by balancing the costs of current reproduction with costs to future survival and fecundity. Males and females typically face different reproductive costs, which makes comparisons of their reproductive strategies difficult. Burying beetles provide a unique system that allows us to compare the costs of reproduction between the sexes because males and females are capable of raising offspring together or alone and carcass preparation and offspring care represent the majority of reproductive costs for both sexes. Because both sexes perform the same functions of carcass preparation and offspring care, we predict that they would experience similar costs and have similar life history patterns. In this study we assess the cost of reproduction in male Nicrophorus orbicollis and compare to patterns observed in females. We compare the reproductive strategies of single males and females that provided pre- and post-hatching parental care. There is a cost to reproduction for both males and females, but the sexes respond to these costs differently. Females match brood size with carcass size, and thus maximize the lifetime number of offspring on a given size carcass. Males cull proportionately more offspring on all carcass sizes, and thus have a lower lifetime number of offspring compared to females. Females exhibit an adaptive reproductive strategy based on resource availability, but male reproductive strategies are not adaptive in relation to resource availability.

Residency time as an indicator of reproductive restraint in male burying beetles.

The cost of reproduction theory posits that there are trade-offs between current and future reproduction because resources that are allocated to current offspring cannot be used for future reproductive opportunities. Two adaptive reproductive strategies have been hypothesized to offset the costs of reproduction and maximize lifetime fitness. The terminal investment hypothesis predicts that as individuals age they will allocate more resources to current reproduction as a response to decreasing residual reproductive value. The reproductive restraint hypotheses predicts that as individuals age they will allocate fewer resources to current reproduction to increase the chance of surviving for an additional reproductive opportunity. In this study, we test for adaptive responses to advancing age in male burying beetles, Nicrophorus orbicollis. Burying beetles use facultative biparental care, but the male typically abandons the brood before the female. Previous work in male burying beetles has suggested several factors to explain variation in male residency time, but no study has observed male behavior throughout their entire reproductive lifetimes to determine whether males change residency time in an adaptive way with age. We compared residency time of males that reproduced biparentally, uniparentally, and on different-sized carcasses to determine if they used an adaptive reproductive strategy. Males did not increase residency time as they aged when reproducing biparentally, but decreased residency time with age when reproducing uniparentally. A decrease in parental care with age is consistent with a reproductive restraint strategy. When female age increased over time, males did not increase their residency time to compensate for deteriorating female condition. To our knowledge, this is the first test of adaptive reproductive allocation strategies in male burying beetles.

Hepatitis C seropositivity is not a risk factor for sensory neuropathy among patients with HIV.

BACKGROUND: Sensory neuropathy (SN) is common in patients with HIV. Hepatitis C (HCV) coinfection is often cited as an HIV-SN risk factor, but data to support this are lacking. This collaboration aimed to examine the association between HCV serostatus and SN risk among ambulatory HIV-positive patients. METHODS: Patients with HIV were assessed in cross-sectional studies in Baltimore, Jakarta, Johannesburg, Kuala Lumpur, Melbourne, and Sydney for SN (defined by both supportive symptoms and signs). HCV seropositivity was assessed as an SN risk using a chi(2) test, followed by logistic regression modeling to correct for treatment exposures and demographics. RESULTS: A total of 837 patients of African, Asian, and Caucasian descent were studied. HCV seroprevalence varied by site (Baltimore n = 104, 61% HCV+; Jakarta 96, 51%; Johannesburg 300, 1%; Kuala Lumpur 97, 10%; Melbourne 206, 16%; Sydney 34, 18%). HCV seropositivity was not associated with increased SN risk at any site, but was associated with reduced SN risk in Melbourne (p = 0.003). On multivariate analyses, the independent associations with SN were increasing age, height, and stavudine exposure. HCV seropositivity was not independently associated with an increased SN risk at any site, but associated independently with reduced SN risk in Baltimore (p = 0.04) and Melbourne (p = 0.06). CONCLUSIONS: Hepatitis C (HCV) seropositivity was not associated with increased sensory neuropathy risk among HIV-positive patients at any site. While we were unable to assess HCV RNA or liver damage, the data suggest that HCV coinfection is not a major contributor to HIV-SN. HCV = hepatitis C; SN = sensory neuropathy.

Cost of reproduction, resource quality, and terminal investment in a burying beetle.

We evaluate the cost-of-reproduction hypothesis in the burying beetle Nicrophorus orbicollis and examine how the importance of this trade-off changes as females age (i.e., the terminal-investment hypothesis). These beetles breed on small vertebrate carcasses, which serve as a food resource for them and their offspring. Consistent with the cost-of-reproduction hypothesis, females manipulated to overproduce offspring suffered a reduction in fecundity and life span when compared to controls, although all reproducing females had reduced life spans compared to nonbreeding females. Older females produced larger broods and allocated less of the carcass to their own body mass and a greater proportion to offspring than did younger females. Resource allocation to offspring increased with age. Females given larger carcasses invested more in current reproduction and less in future reproduction than did females given smaller carcasses. Our results provide unconfounded support for both the cost-of-reproduction hypothesis (i.e., current reproduction constrains future reproductive output) and the terminal-investment hypothesis (i.e., the importance of the trade-off between current and future reproduction declines with age such that allocation to current reproduction should increase as females age).

Elastic moduli of faceted aluminum nitride nanotubes measured by contact resonance atomic force microscopy.

A new methodology for determining the radial elastic modulus of a one-dimensional nanostructure laid on a substrate has been developed. The methodology consists of the combination of contact resonance atomic force microscopy (AFM) with finite element analysis, and we illustrate it for the case of faceted AlN nanotubes with triangular cross-sections. By making precision measurements of the resonance frequencies of the AFM cantilever-probe first in air and then in contact with the AlN nanotubes, we determine the contact stiffness at different locations on the nanotubes, i.e. on edges, inner surfaces, and outer facets. From the contact stiffness we have extracted the indentation modulus and found that this modulus depends strongly on the apex angle of the nanotube, varying from 250 to 400 GPa for indentation on the edges of the nanotubes investigated.

Refinement of interbody implant testing in goats: a surgical and morphometric rationale for selection of a cervical level. Laboratory investigation.

OBJECT: The authors provide a surgical description of the ventral approach to the cervical spine in a goat model and identify selection of the most appropriate level for testing interbody devices. These constructs are designed for implantation in humans during anterior cervical discectomy and fusion. Such description and guidelines for level selection have never been published in either the medical or veterinarian literature. METHODS: The study comprised three phases: surgical, anatomical, and morphometric. Six goats underwent ventral approaches and were later killed; their necks were dissected and the cervical spines were processed to obtain clean specimens of the vertebral bodies. Measurements were made at each level using a contact digitizer. RESULTS: The anterolateral bone spurs, called alar processes, and the increased thickness of the longus colli muscle are the surgically relevant characteristics in the goat. The morphometric analysis showed that C2-3 is the most suitable level for implantation of interbody devices. The vertebral endplates at the C2-3 level are relatively flat and parallel to each other, and are perpendicular to the spinal canal axis. More distally, the endplates adopt a more curved arrangement, and the endplate angle becomes significantly greater than 90 degrees. The authors describe anatomical landmarks that are important to safely and effectively perform a ventral cervical spinal approach in the goat. CONCLUSIONS: The authors' model identifies C2-3 as the most appropriate level for animal testing of cervical implants because of its similarity to human anatomy. Further study with rigorous biomechanical range of motion evaluation of each caprine cervical level is needed.

Antiretroviral use and other risks for HIV-associated neuropathies in an international cohort.

OBJECTIVE: To explore the association between specific nucleoside reverse transcriptase inhibitors and sensory neuropathies (SNs) and define the modifying roles of hepatitis C (HCV), vitamin B12 deficiency, and impaired glucose tolerance. METHODS: The authors conducted a prospective cohort study of 147 HIV-infected adults at two sites chosen to emphasize demographic differences. Standardized assessments included detailed antiretroviral histories, neurologic examinations, skin biopsies for epidermal nerve quantitation, and quantitative sensory testing. RESULTS: There were significant differences between subjects at Johns Hopkins University (JHU) and Monash University (MU) in gender, race, HIV transmission route, and HCV seroprevalence. Symptomatic SN was present in 49% at JHU and 55% at MU (chi2 = 4.02, p = 0.134) and was significantly more common in those at least age 40 than younger patients (odds ratio [OR] = 2.87, 95% CI = 1.27, 6.49). After adjusting for site, age, and CD4 cell count, exposure to didanosine (ddI) or stavudine (d4T) was associated with an significantly increased likelihood of symptomatic SN (ddI: OR = 3.21, 95% CI: 1.56, 6.60; d4T: OR = 7.66, 95% CI: 2.89, 20.33). Plasma HIV RNA, lactate, and HCV were not associated with SN. Quantitative vibratory testing identified neuropathy with a positive predictive value of 76% and epidermal nerve fiber densities 59%. CONCLUSIONS: Exposure to stavudine and didanosine was significantly associated with a heightened risk for symptomatic sensory neuropathy. Reduced vibration thresholds and epidermal nerve fiber densities had the highest diagnostic efficiency of the laboratory indicators of neuropathy examined, but were relatively insensitive in isolation.

Complex formation of trimethylaluminum and trimethylgallium with ammonia: evidence for a hydrogen-bonded adduct.

We have investigated the formation of gas-phase adducts of trimethylaluminum and trimethylgallium with ammonia using room-temperature Fourier transform infrared experiments and density functional theory calculations. Our results indicate for the first time that, at higher partial pressures, a product distinct from the well-known (CH3)3M:NH3 adduct grows in for both M = Al and M = Ga. Comparison of the experimental and calculated IR spectra, along with calculations of the energetics, indicates that this second product is the result of hydrogen bonding of a second NH3 molecule to the (CH3)3M:NH3 adduct and can be written as (CH3)3M:NH3...NH3. The binding energy of this hydrogen-bonded adduct is calculated to be 26.8 kcal/mol for M = Al and 18.4 kcal/mol for M = Ga and is lower in energy (more stable) relative to the 1:1 (CH3)3M:NH3 adduct by 7.2 kcal/mol for M = Al and 6.6 kcal/mol for M = Ga. In contrast, an alternative complex involving the formation of two separate M-N donor-acceptor bonds, which is written as H3N:(CH3)3M:NH3, is calculated to be lower in energy relative to (CH3)3M:NH3 by only 0.1 kcal/mol for M = Al and 0.2 kcal/mol for M = Ga and is not observed experimentally. These results show that hydrogen bonding plays an important role in the interaction of ammonia with metal organic precursors involving Al, Ga, and In, under typical metal organic chemical vapor deposition AlGaInN growth conditions.

Kinetics of metal organic-ammonia adduct decomposition: implications for group-III nitride MOCVD.

We have used infrared spectroscopy to investigate the decomposition of the gas-phase (Me)(3)M:NH(3) (M = Al, Ga, In) adducts from room temperature to 573 K, at reactant concentrations in the nominal range used for Al(Ga,In)N metal organic chemical vapor deposition. At 473-523 K TMAl:NH(3) decomposes quantitatively to yield (Me(2))AlNH(2) and CH(4). Comparison of the experimental and theoretical spectra indicates that the majority of the aluminum metal organic product exists in dimer form, i.e., [(Me(2))AlNH(2)](2). The decomposition reaction exhibits unimolecular decomposition kinetics with rate constant parameters of nu = 1 x 10(12) s(-1) and E(a) = 25.7 kcal/mol. At temperatures <543 K, TMGa + NH(3) and TMIn + NH(3) mixtures are dominated by reversible adduct formation-dissociation with no detectable quantities of CH(4) produced. At 574 K a small amount of decomposition is observed in TMGa + NH(3) mixtures, which can be explained by a simple kinetic model that includes the effect of adduct equilibrium. Results demonstrate that the (Me)(3)Al:NH(3) decomposition rate is fast enough to contribute to the early stages of a concerted parasitic chemical reaction mechanism, but the (Me)(3)Ga:NH(3) decomposition rate is too slow.

Reversible adduct formation of trimethylgallium and trimethylindium with ammonia.

We have used gas-phase infrared spectroscopy to determine the equilibrium constant (K(p)) for the formation of (CH(3))(3)Ga:NH(3) and (CH(3))(3)In:NH(3) adducts in the 80-230 degrees C range. In this temperature range, and at reactant concentrations typically used for metal organic chemical vapor deposition, the dominant chemical reaction is reversible adduct formation/dissociation. Reaction enthalpies and entropies are extracted from the temperature dependence of K(p), yielding DeltaH(Ga) = -16.3 +/- 0.5 kcal/mol, DeltaS(Ga) = -32.4 +/- 1.2 eu, and DeltaH(In) = -15.0 +/- 0.6 kcal/mol, DeltaS(In) = -30.3 +/- 1.4 eu. These results will aid current and future modeling efforts, as well as advance our general understanding of the group-III nitride deposition process.

Mutations of the cationic trypsinogen gene in hereditary and non-hereditary pancreatitis.

BACKGROUND/AIMS: Mutations in the cationic trypsinogen gene have been detected in patients with hereditary pancreatitis (HP). This study investigates the prevalence of the R122H, N29I, A16V and -28delTCC mutations in the common, non-hereditary forms of chronic pancreatitis and in a HP family. METHODS: DNA was prepared from blood samples of 53 patients with chronic pancreatitis (36 alcoholic, 14 idiopathic and 3 hereditary), 20 alcoholic controls and 20 healthy, ethnically matched controls. The R122H and A16V mutations were identified by the polymerase chain reaction (PCR) and restriction enzyme digestion. A nested-PCR was used to identify the N29I mutation. The -28delTCC deletion and the C133807T polymorphism were sought by direct sequencing. RESULTS: The R122H mutation was detected in 1 patient with alcoholic chronic pancreatitis and all 3 affected members of a HP family. The N29I, A16V and -28delTCC mutations were not detected in any of the study subjects. At the C133807T polymorphism, the C allele and C/C genotype were significantly increased in alcoholic chronic pancreatitis (p = 0.001 and p = 0.0004, respectively) while the T allele and CT genotype were significantly reduced (p = 0.001 and p = 0.004, respectively) compared to healthy controls. CONCLUSIONS: Mutations of the cationic trypsinogen gene are rarely found in chronic pancreatitis patients of typical aetiology. Screening for these mutations should be considered in those with a family history consistent with hereditary pancreatitis but may also be appropriate in a well-defined subgroup of patients with non-hereditary chronic pancreatitis, i.e. those who have developed the disease before the age of 30.

Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability.

The present study evaluated the necessity of store-operated Ca(2+) entry in mediating thrombin-induced 20-kDa myosin light chain (MLC(20)) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 microM) activated Ca(2+) entry through a common pathway in confluent BPAECs. Similar increases in MLC(20) phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC(20) phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC(20) phosphorylation when Ca(2+) influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 A) transfer across confluent BPAEC monolayers. Activation of store-operated Ca(2+) entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca(2+) entry is coupled with MLC(20) phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca(2+) entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca(2+) signaling.