Zap1 is required for Candida glabrata response to fluconazole.

The increasing prevalence of fluconazole-resistant clinical isolates of Candida spp. strongly hinders the widespread use of the drug. To tackle this problem, great efforts have been made to fully understand the fungal response to fluconazole. In this work, we show that the role of Zap1 in Candida glabrata goes beyond regulating yeast adaptation to zinc deficiency. In line with our previous observation that deletion of ZAP1 makes yeast cells more sensitive to fluconazole, we found that the mutant CgΔzap1 accumulates higher levels of the drug, which correlates well with its lower levels of ergosterol. Surprisingly, Zap1 is a negative regulator of the drug efflux transporter gene CDR1 and of its regulator, PDR1. The apparent paradox of drug accumulation in cells where genes encoding transporters relevant for drug extrusion are being overexpressed led us to postulate that their activity could be impaired. In agreement, Zap1-depleted cells present, in addition to decreased ergosterol levels, an altered composition of membrane phospholipids, which together should impact membrane function and impair the detoxification of fluconazole. Overall, our study brings to light Zap1 as an important hub in Candida glabrata response to fluconazole.

Recovery balance: a method for estimating losses in a Bacillus anthracis spore sampling protocol.

AIM: The aim of this study was to develop a method to calculate the performance, and isolate error contributions occurring in a microbial surface sampling protocol. METHODS AND RESULTS: The experiments were conducted using a slip/peel tester to provide consistent pressure during the wipe collection. Fluorescence microscopy was used to count spores deposited on the coupon prior to sampling. The mean recovery efficiency (RE) as well as the efficiency of each step in the process was estimated by a recovery balance (RB), similar to a mass balance. Two studies were conducted in this work. In the first one, the recovery of spores from the solution (RE(soln)) was 57.7% (SD = 8.0), while spores left on the glass surface after wiping (RE(b+c)) was 2.8% (SD = 2.4). The RE of spores adhered to the tube wall (RE(tube)) and glass surface (RE(surf)) was 1.2% (SD = 19.6) and 5.8% (SD = 7.1), respectively. From the recovery balance, it was determined that 39.9% (SD = 21.2) of spores were lost to the wipe (RE(wipe)). The applicability of the RB method was demonstrated in a second study by examining the relative impact of parameters affecting spore collection including relative humidity, wipe material, wetting agent and nonporous surfaces. CONCLUSIONS: The approach used in this study pointed out the need for a closer analysis of the complex interaction between spores and wipe material because a substantial percentage of spores were lost to the wipe. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery balance, in association with independent controls, provides an account for error contribution and potential variability on each step of the sampling protocol. The approach is not meant to be a replacement for field or laboratory validation of wipe recoveries but promote the development of new collection methodologies and support protocol optimization in laboratory settings.

Description of Larval Instars To Fill a Gap in Forensic Entomology: The Larvae of Paralucilia pseudolyrcea (Diptera: Calliphoridae).

A fundamental assumption of forensic entomology for estimating the postmortem interval is that insect species are accurately identified, which depends on diagnostic morphological characters. Larvae of the blow fly Paralucilia pseudolyrcea (Mello, 1969) (Diptera: Calliphoridae) were sampled from four corpses in the state of Paraná, Brazil, but despite the forensic importance of this species, morphological data for the identification of its larval instars are lacking, limiting its usefulness in such cases. Thus, the main goal of this study was to describe the larval instars of P. pseudolyrcea. The material was obtained from a colony established by larvae collected from a corpse of a murder case. Overall, the distribution of spines is a key character for identifying this species in the first, second and third instars. Other characteristics, such as the presence of an accessory oral sclerite, the small cirri, the number of lobes of the anterior spiracle and the morphology of posterior spiracles, separates P. pseudolyrcea from other necrophagous blow flies. The detailed morphological description provided here facilitates the identification of larval instars of P. pseudolyrcea and their differentiation from those of other calliphorid species.

Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

AIMS: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). METHODS AND RESULTS: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 10(7) cells ml(-1)), intermediate (4 × 10(5) cells ml(-1)) and low (4 × 10(3) cells ml(-1)), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively. CONCLUSIONS: The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material. SIGNIFICANCE AND IMPACT OF THE STUDY: The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies.

Aided visual inspection with acetic acid (VIA) and HPV detection as optional screening tools for cervical cancer and its precursor lesions.

PURPOSE OF INVESTIGATION: To assess the contribution of visual inspection with acetic acid (VIA) and Hybrid Capture II (HCII) as adjunct methods to the Pap test in detecting cervical neoplasia. SUBJECTS AND METHODS: This was a cross-sectional study with 809 women who consecutively attended gynecological consultations at Campinas University, Brazil, from January 2002 to July 2003. Pap test, HCII, VIA, and colposcopy were offered to all patients. Performance of tests (alone or in combination) in detecting histologically confirmed lesions was evaluated. RESULTS: Of the 40 patients with CIN, 69% had CIN1, 26% CIN2 or CIN3 and one patient had invasive carcinoma. VIA had the best performance in detecting CIN, yielding 72% sensitivity and 91% specificity. Considering only CIN2 or worse as significant lesions, HCII had the best sensitivity (73%), while the Pap test was the most specific (93%). Combining the three exams, 92% of the CIN1 or worse were detected. When CIN1 was excluded from the analysis, Pap smear plus HCII delivered 82% sensitivity and 79% specificity. However, this combination yielded a very low (5%) PPV. CONCLUSION: VIA and HCII contributed to the screening of cervical neoplasia in a group of Brazilian women, but the cost-effectiveness of conjoint screening modalities is still debatable.

Protective effect of a protein-free diet in acute experimental pancreatitis.

1. Survival time, amylase activity and pancreatic protein, DNA and RNA content were measured in 38 control rats and in 104 rats injected with sodium taurocholate to induce acute pancreatitis after 21 days on one of four diets differing in protein, lipid and carbohydrate content. 2. All of the parameters measured were lower in rats with pancreatitis than in controls maintained on the same diets. Among the rats with pancreatitis, those receiving a protein-free diet survived longer and had significantly higher DNA, lower amylase activity and lower RNA and protein levels than those receiving a balanced diet or one that was high in protein or lipid content. 3. We conclude that acute pancreatitis in rats has a more benign course in protein-undernourished animals.

Production of anti-HBsAg monoclonal antibodies.

The production of monoclonal antibodies against the HBsAg is reported. Balb/c mice immunized against a commercial vaccine were used. Upon fusion of spleen cells from an animal having a high titer with the SP2/0 myeloma cell line, we obtained 6 stable cell lines, all of the IgG1 subclass. They showed a wide range of specificities against the classical HBsAg subtypes. These monoclonal antibodies can be used as the basis for the development of new methods for the screening and study of the hepatitis B virus.

[Avidity of specific IgG antibodies as markers of recent primary infection caused by Toxoplasma gondii]. / Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma Gondii.

For serologically characterizing a recent primary toxoplasma infection, the low avidity of IgG specific antibodies was studied. Avidity was evaluated as the decrease of IgG antibody titers in ELISA after treating plates with 6 M urea, as a dissociating solution of low avidity antigen-antibody complexes. Sixty nine serum samples were studied, presenting characteristic patterns of recent, transitional or chronic toxoplasmosis. Serological patterns were determined according to results of IgG and IgM immunofluorescence, IgM-capture, and hemagglutination tests. Twenty three serum samples from each of the referred patterns I, II and III were titrated. For chronic toxoplasmosis infections, which presented a serological pattern III, observed decrease of titers was 3% +/- 3%. For pattern I recent toxoplasmosis sera it was 34% +/- 12%, and for transition pattern II, 12% +/- 9%. Thus, a low avidity of IgG specific antibodies can be applicable for the diagnosis of a recent toxoplasmosis infection.

[Passive microhemagglutination reaction to Treponema pallidum in the cerebrospinal fluid in the diagnosis of neurosyphilis]. / Contribuição da reação de micro-hemaglutinação passiva para o treponema pallidum (mha-tp) no líquido cefalorraqueano ao diagnóstico de neurossífilis.

A quantitative micro-hemagglutination test for antibodies to Treponema pallidum (MHA-TP) was evaluated in 25 cerebrospinal fluid (CSF) samples obtained from patients with neurosyphilis (NS group) and in 7 CSF samples of patients with reactive serologic tests for syphilis (STS+). These data were compared to treponemal and nontreponemal tests. The MHA-TP was reactive in all of the 25 NS group samples, the FTA-Abs and the complement fixation of Wassermann (CFW) were in 24 and the VDRL in only 9. In the 7 STS+ samples (STS+ group), the MHA-TP was reactive in 6, the FTA-Abs in all of them and the nontreponemal tests were nonreactive. Results analyses support conclusion the clinical diagnosis of NS must be complemented by cytoproteic dual and immunological treponemal and nontreponemal assays in CSF. The MHA-TP test was as sensitive as FTA-Abs and required less technical and interpretative skills, contributing in association to CFW to NS diagnosis.

In vitro quantitative evaluation of marginal microleakage in Class II restorations confected with a glass ionomer cement and two composite resins.

This study evaluated, in vitro, marginal microleakage in class II restorations confected with the glass ionomer cement Vitremer and with the composite resins Ariston pHc and P-60. The aims of the study were to assess the effect of thermocycling on those materials and to evaluate two methods utilized in the analysis of dye penetration. Sixty premolars divided in three groups were utilized; the teeth had proximal cavities whose cervical walls were located 1 mm below the cementoenamel junction. Half of the test specimens from each group underwent thermocycling; the other half remained in deionized water, at 37 degrees C. The specimens were immersed, for 24 hours, in a basic 0.5% fuchsin solution at 37 degrees C. For the analysis of microleakage, the specimens were sectioned in a mesio-distal direction, and the observation was carried out with the software Imagetools. The results were evaluated through the 2-way ANOVA and through the Tukey's test. All groups presented marginal microleakage. The smallest values were obtained with Vitremer, followed by those obtained with the composite resins P-60 and Ariston pHc. There was no statistically significant difference caused by thermocycling, and the method of maximum infiltration was the best for detecting the extension of microleakage.

[Assessment of microleakage of adhesive restorative materials in deciduous teeth]. / Avaliação da infiltração marginal de materiais restauradores adesivos em dentes decíduos.

The microleakage of Chelon-Fil, Vitremer, Compoglass-F and Z100 was evaluated in deciduous teeth. Class V restorations were placed in the buccal aspect of 40 extracted primary canines; they were thermocycled, and immersed in 0.5% basic fuchsin. Three slices of each tooth were obtained in order to analyze the penetration of dye in the gingival and occlusal walls, under magnification of 40 X. The results showed that Chelon-Fil had the highest degree of marginal leakage. Vitremer, Compoglass-F and Z100 presented similar penetration of dye. The microleakage of Chelon-Fil and Vitremer was greater in the gingival wall than in the occlusal wall. Microleakage was the same in both gingival and occlusal walls when cavities were filled, either with Z100 or with Compoglass-F.

Soil temperature and diapause maintenance in eggs of the spittlebug, Deois flavopicta (Hemiptera: Cercopidae).

Diapausing eggs of the neotropical pasture pest, Deois flavopicta (Stal) (Hemiptera: Cercopidae), were exposed to low overnight temperatures that simulated field conditions during the dry season (23/12, 23/15 and 23/18 degrees C day/night), for different periods (0-60 days). After treatment, eggs were kept at 28 degrees C and contact water (100% humidity) until hatching. A group of diapausing eggs were kept all the time under this last condition as a control treatment. Time for hatching (in degree-days) was reduced with decrease in low overnight temperature and increase of exposure time to these cold shocks, although there was no interaction between the factors. Regression of exposure time to cold shock influencing the expected mean hatching time produced independent equations for temperatures below 18 degrees C and 15 degrees C. We constructed a model that simulates the expected proportion of the population hatching after the beginning of rainy season based on regression equations to mean hatching time and associated standard deviation. The simulation generated for the model correlated significantly with nymphal population observed in the field. These results showed that overnight soil temperatures below 18 degrees C, as occurs in Central and South-eastern Brazil between May and August, shorten the period of diapause, increase quiescent eggs in the soil, and may have synchronize population hatching.

Ischemic preconditioning and atenolol on lung injury after intestinal ischemia and reperfusion in rats.

The aim of this study was evaluate the beta blocker atenolol (AT) and ischemic preconditioning (IPC) strategies for tissue protection against systemic effects of intestinal ischemia (I) and reperfusion (R) injury. Forty-two rats were pretreated with AT (1.5 mg · kg(-1)), 0.9% saline solution (SS; 0.1 mL), or IPC and then subjected to prolonged occlusion of the superior mesenteric artery for 60 minutes leading to I followed or not by 120 minutes of R, according to the group. For IPC, 5 minutes of I prior to 10 minutes of R were established. After this process of I or I-R, the right lung of each animal was adequately prepared for staining with hematoxylin and eosin and subsequent histologic analysis for quantification of inflammatory infiltrate was done. The left lung was frozen and prepared for assessment of oxidative stress by the quantification of thiobarbituric acid-reactivity substances (TBARS). Histologic analysis showed an important inflammatory infiltrate in the I-R + SS (I-R + SS = 4.5), which was significantly (P < .05) reduced by IPC (I-R + IPC = 3.0) or AT (I-R + AT = 3.0). Likewise, the TBARS levels were decreased by both strategies (I-R + SS = 0.63; I-R + IPC = 0.23; I-R + AT = 0.38; P < .05). Our results showed that AT and IPC attenuate pulmonary lesions caused by intestinal I and R process.

Enhanced peroxisomal ß-oxidation is associated with prevention of obesity and glucose intolerance by fish oil-enriched diets.

OBJECTIVE: The effects of different amounts of omega 3-polyunsaturated fatty acids in diets with normal or high content of fat on lipid and carbohydrate metabolism were investigated. DESIGN AND METHODS: Mice were fed for 8 weeks on diets enriched with fish oil or lard at 10% or 60% of energy. Energy balance and energy expenditure were analyzed. Fatty acid (FA) oxidative capacity of the liver and the activity of enzymes involved in this pathway were assessed. RESULTS: Fish oil-fed mice had lower body weight and adiposity compared with lard-fed animals, despite having lower rates of oxygen consumption. Mice fed diets containing fish oil also displayed lower glycemia, reduced fat content in the liver, and improved glucose tolerance compared with lard-fed animals. The fish oil-containing diets increased markers of hepatic peroxisomal content and increased the generation of metabolites derived from FA ß-oxidation in liver homogenates. In contrast, no changes were observed in the content of mitochondrial electron transport chain proteins or carnitine palmitoyl transferase-1 in the liver, indicating little direct effect of fish oil on mitochondrial metabolism. CONCLUSION: Collectively, our findings suggest that the energy inefficient oxidation of FAs in peroxisomes may be an important mechanism underlying the protection against obesity and glucose intolerance of fish oil administration.

The effect of intravenous insulin infusion on renal blood flow in conscious sheep is partially mediated by nitric oxide but not by prostaglandins.

To test the effect of insulin on renal perfusion and the participation of NO and PG as mediators of this response, renal blood flow (RBF) was measured in sheep (n = 8) implanted with ultrasonic flow probes around renal arteries and with a systemic arterial pressure (SAP, n = 4) telemetry device. Three protocols were performed: 1) RBF and SAP were recorded (0800 to 1800 h) in fed and fasted sheep, with the latter receiving intravenous (i.v.) infusions (0.5 mL/min) of insulin at 2 or 6 mU/(kg·min); 2) fasted sheep received i.v. infusions of either an inhibitor of NO synthesis (N(G)-nitro-L-arginine methyl ester, L-NAME) alone [0.22 mg/(kg·min), 1000 to 1200 h] or L-NAME (1000 to 1200 h) + insulin during the second hour (6 mU/(kg·min), 1100 to 1200 h); and 3) the same protocol was followed as in protocol 2, substituting L-NAME with ketoprofen [0.2 mg/(kg·min)], a cyclooxygenase inhibitor. In all protocols, plasma insulin and glucose were determined. During insulin administration, euglycemia was maintained and hypokalemia was prevented by infusing glucose and KCl solutions. After the onset of meals, a long-lasting 18% increase in RBF and a 48% insulin increase were observed (P < 0.05), without changes in SAP. Low- and high-dose insulin infusions increased RBF by 19 and 40%, respectively (P < 0.05). As after meals, the increases in RBF lasted longer than the insulin increase (P < 0.05). The L-NAME infusion decreased RBF by 15% (P < 0.05); when insulin was added, RBF increased to preinfusion values. Ketoprofen decreased RBF by 9% (P < 0.05); when insulin was added, RBF increased to 13% above preinfusion values (P < 0.05). In no case was a modification in SAP or glucose noted during the RBF changes. In conclusion, insulin infusion mimics the meal-dependent increase in RBF, independent of SAP, and lasts longer than the blood insulin plateau. The RBF increase induced by insulin was only partially prevented by L-NAME. Ketoprofen failed to prevent the insulin-dependent RBF increase. Both facts suggested that complementary vasodilatatory agents accounted for the insulin effect on sheep renal hemodynamics.

Mitochondrial targeting for photochemotherapy. Can selective tumor cell killing be predicted based on n-octanol/water distribution coefficients?

The concept of mitochondrial targeting for chemo- and photochemotherapy of neoplastic diseases has its origin in the observation that enhanced mitochondrial transmembrane potential is a common tumor cell phenotype. As a result of this enhanced transmembrane potential, a number of cationic dyes accumulate in larger amounts and are retained for longer periods in the mitochondria of tumor cells than in normal cells. Only a relatively small number of (photo)toxic dyes known to localize in energized cell mitochondria are capable of inducing the destruction of tumor cells with desirable degrees of selectivity, however. We investigated how lipophilic character may affect the degree of specificity with which cationic dyes localize in energized cell mitochondria and how mitochondrial specificity may affect tumor cell selectivity. To this end, we used fluorescence microscopy to characterize the subcellular localization of ethyl violet and seven analogs of the prototypical mitochondria-specific dye, rhodamine 123. All cationic rhodamines studied here (-0.62 < log D(ow) < 1.60, where D(ow) represents the n-octanol/water distribution coefficient) were found to show considerable mitochondrial specificity, while the more lipophilic ethyl violet (log D(ow) = 2.37) did not. Ethyl violet was found to localize not only in mitochondria, but also in lysosomes. We also compared the degree of selective tumor cell killing induced by ethyl violet and two phototoxic rhodamines, i.e., the dibromo derivatives of rhodamine 123 and its n-octyl ester analog. While ethyl violet induces the destruction of human uterine sarcoma (MES-SA) cells and normal green monkey kidney cells (CV-1) with comparable efficiency, the mitochondria-specific dibromorhodamines were found to induce the destruction of MES-SA cells with considerable selectivity. Our findings are consistent with the premise that mitochondrial localization per se does not provide successful selective tumor cell killing using mitochondrial targeting. Our results reinforce the hypothesis that while most cationic dyes can be expected to localize at least to some extent in energized cell mitochondria, only those showing virtually absolute mitochondrial specificity can actually mediate the destruction of tumor cells with desirable selectivity. These findings also support the hypothesis that the probability of success of mitochondrial targeting in photochemotherapy of neoplastic diseases is bound to be higher when the D(ow) associated with the drug candidate falls within approximately two orders of magnitude of that of rhodamine 123.

Late infantile autosomal recessive myotonia, mental retardation, and skeletal abnormalities: a new autosomal recessive syndrome.

Four sibs of non-consanguineous parents who had myotonia from late infancy are described. Mild to moderate mental retardation, severe bone abnormalities of the vertebral column (mainly in the thoracolumbar region), and short stature were also observed. Autosomal recessive inheritance is demonstrated. These cases are compared with reported cases of the Schwartz-Jampel syndrome.

Isolation and characterization of collagen messenger RNA*.

Chick embryo collagen-synthesizing polysomes were isolated by differential centrifugation. RNA extracted from these particles was chromatographed in oligo(dT)-cellulose solumns and the mRNA thus obtained characterized as collagen mRNA by its electrophoetical mobility in acrylamide gels (equivalent to 1.05 x 10-6 daltons) and its effect upon a cell-free system derived from Krebs ascites tumor cells. The incorporation of 3H-proline was markedly dependent upon rabbit reticulocyte initiation factors and inhibited by initiation inhibitors such as aurintricaboxilate and pyrocatechol violet. The incorporation product was characterized as collagen by its lack of tryptophan, digestibility by purified bacterial collagenase, and by its co-chromatography with unlabled chick collagen in Sephadex G-200 and CM-cellulose columns.