Historical Pollution of an Urban Watershed Based in Geochemical, Geoacumulation, and EROD Activity in PLHC-1 Analyses in Sediment Cores.

In the present study, the environmental changes over the past 100 years in the Cambé River watershed in Southern Brazil were investigated. For this, a sediment core was collected to examine the distribution of metals, polycyclic aromatic hydrocarbons (PAHs), and ecotoxicological markers. The core corresponds from 1914 to 2012, which was obtained by the decay of 210Pb. Changes in the study area also were examined by: Geoaccumulation index (Igeo) and enrichment factor (EF), sediment quality guidelines (SGQs), and ethoxyresorufin-O-deethylase (EROD) activity in Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cells line. The Σ16 PAHs ranged from 242.6 to 40,775 ng g-1, with pyrogenic source at the beginning of the core, which likely corresponds to the burning of forests to establish the city and the later use of fossil fuels from 1960 to 2012. In the 1930s, most of metals presented a concentration below the background. After the 1930s, high concentrations can be associated with the intense use of fertilizers (Cd, Cu, Zn, Hg) and increases in urban traffic (Zn and Pb). Igeo showed that the distribution of Cu is considered moderately to strongly polluted, and the EF of Pb was considered as moderately polluted. Statistical analyses showed a strong relation between the presence of the ANP, FLU, PHE, BaP, IND, and Bghi compounds and the induction of EROD activity, and no correlation with heavy metals. A prediction model for the linear regression was obtained between the ANP and BaP concentrations and the EROD activity, with an accuracy of 99%.

Elastogram quality assessment score in vibration-controlled transient elastography: Diagnostic performance compared to digital morphometric analysis of liver biopsy in chronic hepatitis C.

Vibration-controlled transient elastography (VCTE) is widely used for noninvasive fibrosis staging in chronic hepatitis C. However, internal validation is based solely on variability and success rate and lacks reproducible quality indicators. We analysed the graphic representation of shear wave propagation in comparison with morphometric results of liver biopsy, eliminating observer variability bias. Individual elastograms were classified according to two morphologic criteria: extension of wave propagation (length of the graphic representation) and shear wave dispersal (level of parallelism displayed in the elastogram). Then, a score based on these criteria stratified the elastogram in classes I through III (highest to lowest technical quality). Liver stiffness results of each measurement were compared with collagen contents in liver biopsy by morphometric analysis. A total of 3243 elastograms were studied (316 patients). Digital morphometry in liver biopsy showed significant fibrosis in 66% of samples and advanced fibrosis in 31%. Elastogram quality analysis resulted in 1438 class I measurements (44%), 1070 class II (34%) and 735 class III. Area under the receiver operating curve (AUROC) for severe fibrosis according to class (I, II and III) was 0.941, 0.887 and 0.766, respectively. For advanced fibrosis, AUROCs were 0.977, 0.883 and 0.781, respectively. Spearman's correlation testing for all classes and levels of fibrosis demonstrated significant independent association (r2  = -.95, P < .01). Our study is the first to propose measurable quality criteria for VTCE and to validate them against objective assessment of liver biopsy through digital morphometric imaging analysis. We concluded that VCTE performance is significantly influenced by quality assessment of individual measurements. Considering these criteria in clinical practice may improve accuracy.

Beneficial use of industrial by-products for phytoremediation of an arsenic-rich soil from a gold mining area.

This study investigated two industrial by-products - red mud (RM) and its mixture with phosphogypsum (RMG), as amendments in an As((5+))-contaminated soil from a gold mining area in Brazil in order to grow three plant species: Brachiaria decumbens, Crotalaria spectabilis, and Stylosanthes cv. Campo Grande. These amendments were applied to reach a soil pH of 6.0. Using RM and RMG increased shoot dry matter (SDM) and root dry matter (RDM) of most plants, with RMG being more effective. Adding RMG increased the SDM of Brachiaria and Crotalaria by 18 and 25% and the RDM by 25 and 12%, respectively. Stylosanthes was sensitive to As toxicity and grew poorly in all treatments. Arsenic concentration in shoots of Brachiaria and Crotalaria decreased by 26% with the use of RMG while As in roots reduced by 11 and 30%, respectively. Also, the activities of the plant oxidative stress enzymes varied following treatments with the by-products. The plants grew in the As-contaminated soil from the gold mining area. Thus, they might be employed for phytoremediation purposes, especially with the use of RMG due to its potential advantage in terms of nutrient supply (Ca(2+) and SO4(2-) from phosphogypsum).

Safety and on-treatment efficacy of telaprevir: the early access programme for patients with advanced hepatitis C.

BACKGROUND AND AIM: Severe adverse events (AEs) compromise the outcome of direct antiviral agent-based treatment in patients with advanced liver fibrosis due to HCV infection. HEP3002 is an ongoing multinational programme to evaluate safety and efficacy of telaprevir (TVR) plus pegylated-interferon-α (PEG-IFNα) and ribavirin (RBV) in patients with advanced liver fibrosis caused by HCV genotype 1 (HCV-1). METHODS: 1782 patients with HCV-1 and bridging fibrosis or compensated cirrhosis were prospectively recruited from 16 countries worldwide, and treated with 12 weeks of TVR plus PEG-IFN/RBV, followed by 12 or 36 weeks of PEG-IFN and RBV (PR) alone dependent on virological response to treatment and previous response type. RESULTS: 1587 patients completed 12 weeks of triple therapy and 4 weeks of PR tail (53% cirrhosis, 22% HCV-1a). By week 12, HCV RNA was undetectable in 85% of naives, 88% of relapsers, 80% of partial responders and 72% of null responders. Overall, 931 patients (59%) developed grade 1-4 anaemia (grade 3/4 in 31%), 630 (40%) dose reduced RBV, 332 (21%) received erythropoietin and 157 (10%) were transfused. Age and female gender were the strongest predictors of anaemia. 64 patients (4%) developed a grade 3/4 rash. Discontinuation of TVR due to AEs was necessary in 193 patients (12%). Seven patients died (0.4%, six had cirrhosis). CONCLUSIONS: In compensated patients with advanced fibrosis due to HCV-1, triple therapy with TVR led to satisfactory rates of safety, tolerability and on-treatment virological response with adequate managements of AEs.

Citric Acid Conditioning as an Alternative to EDTA for Growth Factors Release and Stem Cell Response in Regenerative Endodontics: A Systematic Review of In Vitro Studies.

INTRODUCTION: Citric acid (CA) conditioning may be a promising alternative to ethylenediaminetetraacetic acid (EDTA) in regenerative endodontic procedures, as reported to improve growth factors' release from dentin. This review systematically investigated the effect of CA conditioning on the growth factors release from dentin and cell behavior compared to EDTA conditioning. METHODS: Searches were conducted (PubMed/MEDLINE, Scopus, Web of Science, Embase, SciELO, Cochrane Library, and grey literature) until May-2023. Only in vitro studies that evaluated the effects of CA on growth factors' release from dentin and cell behavior outcomes compared to EDTA were included. The studies were critically appraised using a modified Joanna Briggs Institute's checklist. Meta-analysis was unfeasible. RESULTS: Out of the 335 articles screened, nine were included. Among these, three studies used dentin discs/roots from permanent human teeth; the rest combined them with stem cells. 10% CA for 5 or 10 minute was the most used protocol. Meanwhile, EDTA concentrations ranged from 10% to 17%. In eight studies examining the release of growth factors, five reported a significant release of transforming growth factor-ß after dentin conditioning with 10% CA compared to 17% EDTA. Regarding cell behavior (6 studies), three studies assessed cell viability. The findings revealed that 10% CA conditioning showed cell viability similar to those of 17% EDTA. Additionally, in two out of three studies, it was observed that 10% CA conditioning did not affect cell morphology. The studies had a low risk of bias. CONCLUSIONS: The use of 10% CA to condition dentin for 5-10 minutes resulted in a notable transforming growth factor -ß1 release, but its cell responses were similar to those of EDTA.

The influence of Amazon River connectivity to littoral meanders on long-term carbon accumulation: A case study of Lake Yahuarcaca.

The objective of this investigation is to evaluate the recent changes in the accumulation of organic matter and carbon on the Yahuarcaca lake system, by means of a multiproxy paleolimnological study. The methodology based on lithological descriptions of 210Pb/137Cs-dated cores allowed us to infer the centennial sedimentation processes and carbon accumulation rates. Sedimentary facies, grain size, magnetic susceptibility, loss on ignition, carbonate, chlorophyll derivatives, stable isotopes of δ13C/δ15N, and carbon accumulation rate were analyzed. LANDSAT and photographic record of satellite images were used to reconstruct the historical geomorphological evolution of the Lake. Sediment cores yielded basal ages of 1827 and 1828 Common Era, representing the formation of lakes as a consequence of the Amazon meandering process. Two main paleolimnological stages were identified, with a boundary transition set at 1980-1984 Common Era, attributed to the geomorphological closure and complete lake separation from the Amazon and the onset of full lentic conditions. This inference was mainly based on both sharp increases in the sedimentation rate from 0.2 to >1 cm yr-1 and carbon accumulation that increased seven-fold (from 2 to 14 g m-2 yr-1) from 1980 to 1984 Common Era. The flood-pulse and connection to the Amazon defined the magnitude of organic inputs, where areas more distant/isolated from the river showed higher accumulation of carbon from autochthonous production, with an average of 8.9 % and 1.10 g m-2 yr-1 (carbon accumulation rate). Those areas closer and connected to the river were strongly related to the interannual hydrological variability, with a lower mean carbon content (5.9 %) and 0.73 g m-2 yr-1 (carbon accumulation rate). We concluded that carbon burial was highest within the most distant spot from the Amazon River because of the weaker connection to the river itself and the more stable lentic conditions for net sedimentation.

The effect of agricultural intensification and water-locking on the world's largest coastal lagoonal system.

The palaeolimnological conditions of Mirim Lagoon, a large coastal shallow lagoon under the influence of historical human impacts related to the development of the primary sector of the economy were reconstructed. The first significant human impact consisted of locking the estuarine system to induce the transition from brackish to freshwater conditions. During this transition, the sedimentation rate consistently increased from pre-disturbance values of 0.25 cm yr-1 to >1 cm yr-1. A concomitant increase in nitrogen and carbon values was recorded indicating a related eutrophication process. The highest nutrient levels were achieved during the 1990s after the incorporation of cutting-edge technologies for agricultural production such as high-yielding varieties of rice resistant to climate variability and pests, and the use of inorganic fertilisers, pesticides and water supply controlled by irrigation. After 2011, the soybean production boosted and the area cultivated with this oilseed equalled the area of rice paddies, i.e., 2 × 105 ha. A sharp decrease in δ13C from -19 to -24‰ and in δ15N from 6 to 2‰ were observed in the sedimentary record, indicating a major shift in the composition of the organic matter after the agricultural intensification. Trace elements Cr, Cu, Ni and Zn showed a high positive correlation with Al and Fe, and enrichment factors near 1, indicating a natural and terrigenous source of these elements and also unpolluted conditions. However, the increase of As after 1990 and the positive correlation with Pb was associated with agricultural practices. All elemental ratios (K/Al, Ti/Al and V/Cr) showed constant pre-disturbance trends and a turning point ca. the 1990s. Microplastics were detected from the beginning of the 1990s and increased towards recent sediments, thus corroborating an anthropogenically impacted scenario. Therefore, the development of the primary sector of the economy exerted clear impacts on the environmental quality of the system.

Historical economic and environmental policies influencing trace metal inputs in Montevideo Bay, Río de la Plata.

Montevideo Bay is located in the middle zone of the Rio de la Plata, and since the foundation of the city, several key economic and environmental policies affected the industry, and thus, metal inputs into this ecosystem. The aim of this study is to evaluate the sedimentary geochemical record of Montevideo Bay, in order to determine the historical inputs of anthropogenic metals to the system. In addition, environmental and economic policies of the country were taken into account to infer the relationship between them and the historic metal input. Concentrations of aluminum, chromium, copper, lead, scandium and zinc were analyzed and the EF and SPI indices were calculated. The analysis showed that since Montevideo foundation, metal concentrations increased in accordance with industry development, and the indices as well as the metal concentration represent a reliable footprint of the history of different economic and environmental policies influencing historical industrial activities.

Transient elastography and APRI score: looking at false positives and false negatives. Diagnostic performance and association to fibrosis staging in chronic hepatitis C.

Although long regarded as the gold standard for liver fibrosis staging in chronic hepatitis C (CHC), liver biopsy (LB) implies both the risk of an invasive procedure and significant variability. The aim of this study was to evaluate the diagnostic performance for transient elastography (TE) and aspartate aminotransferase to platelet index (APRI) used alone and in combination compared to liver biopsy and to analyze false positive/negative results. Patients with CHC, and no previous clinical diagnosis of cirrhosis were enrolled to undergo liver biopsy, TE and APRI. A total of 182 adult patients with a median age of 55 years and median body mass index of 26.71 kg/m2 were analyzed. On LB, 56% of patients had significant levels of fibrosis (METAVIR F≥2) and 28% had advanced fibrosis (F3/F4). The strongest performance for both tests was observed for exclusion of advanced fibrosis with good negative predictive values (89 and 86%, respectively). Low necroinflammatory activity on LB was associated with false negative TE. False positives were associated with NASH and smaller LB fragments. Correlation between APRI and Fibroscan for F≥2 was 100% and 84% for F≥3 and remained high in both false negative and false positive instances, correctly identifying F<2 in 71% of cases and F<3 in 78% (and potentially foregoing up to 84% of LB). We concluded that low individual performance indicators could be attributable to limitations of LB. Poorer differentiation of lower levels of fibrosis is a known issue for LB and remains so for noninvasive tests. Good predictability is possible, however, for advanced fibrosis.

Nodule development on the tropical legume Sesbania virgata under flooded and non-flooded conditions.

The interaction between the Brazilian pioneer legume Sesbania virgata and its microsymbiont Azorhizobium doebereinerae leads to the formation of nitrogen-fixing nodules on roots that grow either in well-aerated soils or in wetlands. We studied the initiation and development of nodules under these alternative conditions. To this end, light and fluorescence microscopy were used to follow the bacterial colonisation and invasion into the host and, by means of transmission electron microscopy, we could observe the intracellular entry. Under hydroponic conditions, intercellular invasion took place at lateral root bases and mature nodules were round and determinate. However, on roots grown in vermiculite that allows aerated growth, bacteria also entered via root hair invasion and nodules were both of the determinate and indeterminate type. Such versatility in entry and developmental plasticity, as previously described in Sesbania rostrata, enables efficient nodulation in both dry and wet environments and are an important adaptive feature of this group of semi-tropical plants that grow in temporarily flooded habitats.

Selective loss of RPGRIP1-dependent ciliary targeting of NPHP4, RPGR and SDCCAG8 underlies the degeneration of photoreceptor neurons.

The retinitis pigmentosa GTPase regulator (RPGR) and nephrocystin-4 (NPHP4) comprise two key partners of the assembly complex of the RPGR-interacting protein 1 (RPGRIP1). Mutations in RPGR and NPHP4 are linked to severe multisystemic diseases with strong retinal involvement of photoreceptor neurons, whereas those in RPGRIP1 cause the fulminant photoreceptor dystrophy, Leber congenital amaurosis (LCA). Further, mutations in Rpgrip1 and Nphp4 suppress the elaboration of the outer segment compartment of photoreceptor neurons by elusive mechanisms, the understanding of which has critical implications in uncovering the pathogenesis of syndromic retinal dystrophies. Here we show RPGRIP1 localizes to the photoreceptor connecting cilium (CC) distally to the centriole/basal body marker, centrin-2 and the ciliary marker, acetylated-α-tubulin. NPHP4 abuts proximally RPGRIP1, RPGR and the serologically defined colon cancer antigen-8 (SDCCAG8), a protein thought to partake in the RPGRIP1 interactome and implicated also in retinal-renal ciliopathies. Ultrastructurally, RPGRIP1 localizes exclusively throughout the photoreceptor CC and Rpgrip1(nmf247) photoreceptors present shorter cilia with a ruffled membrane. Strikingly, Rpgrip1(nmf247) mice without RPGRIP1 expression lack NPHP4 and RPGR in photoreceptor cilia, whereas the SDCCAG8 and acetylated-α-tubulin ciliary localizations are strongly decreased, even though the NPHP4 and SDCCAG8 expression levels are unaffected and those of acetylated-α-tubulin and γ-tubulin are upregulated. Further, RPGRIP1 loss in photoreceptors shifts the subcellular partitioning of SDCCAG8 and NPHP4 to the membrane fraction associated to the endoplasmic reticulum. Conversely, the ciliary localization of these proteins is unaffected in glomeruli or tubular kidney cells of Rpgrip1(nmf247), but NPHP4 is downregulated developmentally and selectively in kidney cortex. Hence, RPGRIP1 presents cell type-dependent pathological effects crucial to the ciliary targeting and subcellular partitioning of NPHP4, RPGR and SDCCAG8, and acetylation of ciliary α-tubulin or its ciliary targeting, selectively in photoreceptors, but not kidney cells, and these pathological effects underlie photoreceptor degeneration and LCA.

Transient elastography and APRI score: looking at false positives and false negatives. Diagnostic performance and association to fibrosis staging in chronic hepatitis C

Although long regarded as the gold standard for liver fibrosis staging in chronic hepatitis C (CHC), liver biopsy (LB) implies both the risk of an invasive procedure and significant variability. The aim of this study was to evaluate the diagnostic performance for transient elastography (TE) and aspartate aminotransferase to platelet index (APRI) used alone and in combination compared to liver biopsy and to analyze false positive/negative results. Patients with CHC, and no previous clinical diagnosis of cirrhosis were enrolled to undergo liver biopsy, TE and APRI. A total of 182 adult patients with a median age of 55 years and median body mass index of 26.71 kg/m2 were analyzed. On LB, 56% of patients had significant levels of fibrosis (METAVIR F≥2) and 28% had advanced fibrosis (F3/F4). The strongest performance for both tests was observed for exclusion of advanced fibrosis with good negative predictive values (89 and 86%, respectively). Low necroinflammatory activity on LB was associated with false negative TE. False positives were associated with NASH and smaller LB fragments. Correlation between APRI and Fibroscan for F≥2 was 100% and 84% for F≥3 and remained high in both false negative and false positive instances, correctly identifying F<2 in 71% of cases and F<3 in 78% (and potentially foregoing up to 84% of LB). We concluded that low individual performance indicators could be attributable to limitations of LB. Poorer differentiation of lower levels of fibrosis is a known issue for LB and remains so for noninvasive tests. Good predictability is possible, however, for advanced fibrosis.

Barriers to treatment of hepatitis C in HIV/HCV coinfected adults in Brazil.

UNLABELLED: The objective of this study was to assess the prevalence of barriers to interferon treatment in a population of HIV/HCV coinfected patients. A cross-sectional study was conducted at two AIDS Outpatient Clinics in Brazil. The study included all HIV infected patients followed at these institutions from January 2005 to November 2007. Medical records of 2,024 HIV-infected patients were evaluated. The prevalence of anti-HCV positive patients among them was 16.7%. Medical records of HCV/HIV coinfected patients were analyzed. 189 patients with the following characteristics were included in our study: mean age 43 years; male gender 65%; former IDUs (52%); HCV genotype 1 (66.4%); HCV genotype 3 (30.5%); median CD4+ T cell count was 340 cells/mm³. Among 189 patients included in the analyses, only 75 (39.6%) were considered eligible for HCV treatment. The most frequent reasons for non-treatment were: non-compliance during clinical follow-up (31.4%), advanced HIV disease (21.9%), excessive alcohol consumption or active drug use (18.7%), and psychiatric disorders (10.1%). CONCLUSIONS: In Brazil, as in elsewhere, more than half of HIV/HCV coinfected patients (60.4%) have been considered not candidates to received anti-HCV treatment. The main reasons may be deemed questionable: non-adherence, drug abuse, and psychiatric disease. Our results highlight the importance of multidisciplinary teams to optimize the access of coinfected patients to HCV treatment.

Haploinsufficiency of RanBP2 is neuroprotective against light-elicited and age-dependent degeneration of photoreceptor neurons.

Prolonged light exposure is a determinant factor in inducing neurodegeneration of photoreceptors by apoptosis. Yet, the molecular bases of the pathways and components triggering this cell death event are elusive. Here, we reveal a prominent age-dependent increase in the susceptibility of photoreceptor neurons to undergo apoptosis under light in a mouse model. This is accompanied by light-induced subcellular changes of photoreceptors, such as dilation of the disks at the tip of the outer segments, prominent vesiculation of nascent disks, and autophagy of mitochondria into large multilamellar bodies. Notably, haploinsufficiency of Ran-binding protein-2 (RanBP2) suppresses apoptosis and most facets of membrane dysgenesis observed with age upon light-elicited stress. RanBP2 haploinsufficiency promotes decreased levels of free fatty acids in the retina independent of light exposure and turns the mice refractory to weight gain on a high-fat diet, whereas light promotes an increase in hydrogen peroxide regardless of the genotype. These studies demonstrate the presence of age-dependent and RanBP2-mediated pathways modulating membrane biogenesis of the outer segments and light-elicited neurodegeneration of photoreceptors. Furthermore, the findings support a mechanism whereby the RanBP2-dependent production of free fatty acids, metabolites thereof or the modulation of a cofactor dependent on any of these, promote apoptosis of photoreceptors in concert with the light-stimulated production of reactive oxygen species.

Acute and subacute toxicity of Schinus terebinthifolius bark extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Schinus terebinthifolius Raddi (Anacardiaceae) has long been used in traditional Brazilian medicine, especially to treat inflammatory and haemostatic diseases. AIM OF THE STUDY: The objective of this study was to evaluate the acute and subacute toxicity (45 days) of Schinus terebinthifolius via the oral route in Wistar rats of both sexes. MATERIALS AND METHODS: For the acute toxicity test, the dried extract of Schinus terebinthifolius bark was administered in doses from 0.625 to 5.0 g/kg (n=5/group/sex) and in the subacute toxicity test the following doses were used: 0.25, 0.625 and 1.5625 g/kg/day (n=13/group/sex), for 45 consecutive days. RESULTS: In the acute toxicity test, Schinus terebinthifolius did not produce any toxic signs or deaths. The subacute treatment with Schinus terebinthifolius did not alter either the body weight gain or the food and water consumption. The hematological and biochemical analysis did not show significant differences in any of the parameters examined in female or male groups, except in two male groups, in which the treatment with Schinus terebinthifolius (0.25 and 0.625 g/kg) induced an increase of mean corpuscular volume values (2.9 and 2.6%, respectively). These variations are within the physiological limits described for the specie and does not have clinical relevance. CONCLUSION: The acute and subacute administration of the dried extract of Schinus terebinthifolius bark did not produced toxic effects in Wistar rats.

Toxicological reproductive study of Cassia occidentalis L. in female Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cassia occidentalis L. (Leguminosae) has long been used as natural medicine in rainforests and other tropical regions for the treatment of inflammation, fever, liver disorders, constipation, worms, fungal infections, ulcers, respiratory infections, snakebite and as a potent abortifacient. AIM OF THE STUDY: This study has investigated the effects of oral sub-acute administration of Cassia occidentalis during pregnancy in female Wistar rats. MATERIALS AND METHODS: Three groups of pregnant rats were treated orally from the 1st to the 6th day (pre-implantation period) and from the 7th to the 14th day (organogenic period) of pregnancy, with doses of 250 and 500 mg/kg. On the 20th day of pregnancy, the animals were euthanized and reproductive parameters evaluated. RESULTS: The results revealed no statistically significant differences between the control and treated groups in terms of offspring/dam relationship; fetuses, placentae and ovaries weights; number of implantation and resorption sites; number of corpora lutea in the ovaries and pre- and post-implantation loss rates. However, the presence of dead fetuses was registered in both doses of 250 and 500 mg/kg of Cassia occidentalis. CONCLUSIONS: Further studies should therefore be conducted to obtain more detailed characteristics of the toxic effects of this species, the use of which is not recommended during pregnancy.

Bovine phospholipase C highly homologous to the norpA protein of Drosophila is expressed specifically in cones.

The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Recently, we reported the isolation and characterization of bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina and with higher homology to the NorpA protein than to any other known PI-PLC. Here, we present evidence that the norpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase.