Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.     

Alternaria alternata prevalence in cereal grains and soybean seeds from Entre Ríos, Argentina

A mycological survey was carried out at Entre Ríos province, Argentina, on sorghum grain, maize, rice, soybean seeds and on freshly harvested and stored wheat. The isolation frequencies and relative densities of species belonging to genera Alternaria, Aspergillus, Fusarium, Penicillium and other fungi were calculated. Alternaria alternata was the major fungal species isolated from sorghum, rice, soybean seeds and on freshly harvested wheat, and a low incidence of Fusarium species was observed on the same substrates. In maize the major fungal species isolated was Fusarium verticillioides. The high incidence levels of A. alternata observed,suggest that it may be necessary to determine, among other mycotoxins, if Alternaria toxins occur in these commodities.     

Role of insulin preincubation in the contractile reactivity of rat aortic rings.

Preincubation with physiological concentrations of insulin affects contractile reactivity of isolated smooth muscle cells. We studied the effects of insulin on intact aortic rings of Wistar rats preincubated 1-2 h with 240 pM (I1) and 960 pM (I2) insulin with and without NO synthesis inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME). Resting force was tripled by 0.1 mM L-NAME in control (C) and I1 groups, but not in I2 groups. I1 treatment decreased the tachyphylaxis to two successive 1 microM arginine vasopressin (AVP) stimulations. Single contractions elicited by 1 microM AVP, 1 microM angiotensin II (AngII), or 0.01 microM endothelin (ET1) were not affected by insulin preincubation in either maximal force (Fmax) or relaxation times. L-NAME enhanced Fmax of AngII contractions by about 75% in C, 120% in I1, and 74% in I2 groups; accordingly, it augmented the final steady-state force in C and I1 but not in I2. Similarly, L-NAME increased Fmax (30-40%) of AVP and ET1 contractions in C and I1 groups but failed to do so in contractions of I2 group. Results obtained with 10 microM indomethacin suggest that this is due to insulin stimulation of prostacyclin effects.     

Homogeneous time-resolved fluorescence assay for identifying p53 interactions with its protein partners,directly in a cellular extract

The p53 protein is a tumor suppressor that protects the organism against malignant consequences of DNA damage. Interaction of p53 with numerous cellular or viral proteins regulates its functional activity either positively or negatively. An approach leading to identification of such protein interactions directly in a cell extract could be of help in the development of screening assays to search for drugs acting on p53 in its cellular environment, either by disrupting its association with inhibitory proteins or by increasing its affinity for activating proteins. We show that the homogeneous time-resolved fluorescence (HTRF) assay based on the time-resolved amplified cryptate emission (TRACE) technology allows identification of such an interaction by simply adding a mixture of two labeled monoclonal antibodies, directly in a cellular extract. We validate this assay by studying p53/SV40-LTAg interactions. The antibodies directed against genuine p53 and SV40-LTAg epitopes were labeled with europium cryptate (donor) and XL665, a crosslinked allophycocyanin (acceptor), respectively. We demonstrated that a nonradiative energy transfer occurs between labeled antibodies only when p53 interacts with SV40-LTag, which opens up the possibility of extending this approach to other p53 partners to search for drugs that restore p53 tumor-suppressor activity.     

Performance of the AutoPap Primary Screening System in the detection of high-risk cases in cervicovaginal smears

OBJECTIVE: To assess the performance of the AutoPap Primary Screening System (APSS) (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) for the detection of high grade cervical squamous intraepithelial lesions and invasive cervical cancer. STUDY DESIGN: A total of 14,779 consecutive conventional Pap smears were processed by the APSS. All slides designated as "Review" by the device were manually screened according to the Bethesda System. The ranking scores obtained from the device were compared with the cytologic interpretations in all cases and with the final histologic diagnoses in the cases with cytologic severe abnormalities. RESULTS: The device classified 10,349 slides as Review (78%) and 2,912 (22%) as "No Further Review." In the 78% Review cases, the samples were ranked in descending order of potential abnormality, broken into quintiles. The correlation between the slide quintile ranks and the manual cytologic diagnosis indicated that 90% of abnormal smears were categorized by the device as in the first and second quintile rank, and the correlation between the rank report of the device and the histologic diagnosis showed that all cases of HSIL or invasive carcinoma were in the top two ranks. No significant abnormalities were observed in any of the smears categorized as No Further Review. CONCLUSION: This study confirmed the effectiveness of APSS for the detection of Pap smears with severe abnormalities.     

Interaction between murine spf-ash mutation and genetic background yields different metabolic phenotypes

The spf-ash mutation in mice results in reduced hepatic and intestinal ornithine transcarbamylase. However, a reduction in enzyme activity only translates in reduced ureagenesis and hyperammonemia when an unbalanced nitrogen load is imposed. Six-week-old wild-type control and spf-ash mutant male mice from different genetic backgrounds (B6 and ICR) were infused intravenously with [(13)C(18)O]urea, l-[(15)N(2)]arginine, l-[5,5 D(2)]ornithine, l-[6-(13)C, 4,4,5,5, D(4)]citrulline, and l-[ring-D(5)]phenylalanine to investigate the interaction between genetic background and spf-ash mutation on ureagenesis, arginine metabolism, and nitric oxide production. ICR(spf-ash) mice maintained ureagenesis (5.5 +/- 0.3 mmol.kg(-1).h(-1)) and developed mild hyperammonemia (145 +/- 19 micromol/l) when an unbalanced nitrogen load was imposed; however, B6(spf-ash) mice became hyperammonemic (671 +/- 15 micromol/l) due to compromised ureagenesis (3.4 +/- 0.1 mmol.kg(-1).h(-1)). Ornithine supplementation restored ureagenesis and mitigated hyperammonemia. A reduction in citrulline entry rate was observed due to the mutation in both genetic backgrounds (wild-type: 128, spf-ash: 60; SE 4.0 micromol.kg(-1).h(-1)). Arginine entry rate was only reduced in B6(spf-ash) mice (B6(spf-ash): 332, ICR(spf-ash): 453; SE 20.6 micromol.kg(-1).h(-1)). Genetic background and mutation had an effect on nitric oxide production (B6: 3.4, B6(spf-ash): 2.8, ICR: 9.0, ICR(spf-ash): 4.6, SE 0.7 micromol.kg(-1).h(-1)). Protein breakdown was the main source of arginine during the postabsorptive state and was higher in ICR(spf-ash) than in B6(spf-ash) mice (phenylalanine entry rate 479 and 327, respectively; SE 18 micromol.kg(-1).h(-1)). Our results highlight the importance of the interaction between mutation and genetic background on ureagenesis, arginine metabolism, and nitric oxide production. These observations help explain the wide phenotypic variation of ornithine transcarbamylase deficiency in the human population.     

Indoor and outdoor airborne fungal propagule concentrations in Mexico City

Thirty homes of asthmatic adults located in Mexico City were examined to determine the predominant culturable fungi and the changes in their airborne concentrations. Fungi were cultured and identified microscopically from air samples collected in naturally ventilated homes, during both wet (July–August) and cool dry (November–December) seasons, and from settled dust from the same homes. Airborne dust from indoor yielded 99–4950 cfu m⁻³, and settled dust 10²–10⁶ cfu g⁻¹ on DG18 agar. The indoor geometric mean concentration of airborne fungi during the cool dry season was 460 cfu m⁻³ while in the wet season it was 141 cfu m⁻³. Similarly, numbers of airborne fungal propagules out of doors decreased 60% between the dry and wet season. In general, the total fungal concentrations in indoor air were less than 10³ cfu m⁻³ and a large proportion of them was collected in Stage-2 of the Andersen sampler. Moreover, the ratio between indoor and outdoor concentrations was <3:1. Five of the 30 sampled homes yielded >500 cfu m⁻³ of one genus, with up to 1493Cladosporium cfu m⁻³ or 2549Penicillium cfu m⁻³. Also, these two genera were predominant in both airborne and settled dust, and their concentrations were greater indoors than out, indicating a possible indoor source of fungal propagules. The predominant species wereCladosporium herbarum, Penicillium aurantiogriseum andP. chrysogenum. These results suggest that exposure to large concentrations of fungi occurs indoors and is associated with both seasons of the year and with particular home characteristics.     

Predictive mathematical modeling of knee static laxity after ACL reconstruction: in vivo analysis

Previous studies did not take into consideration such large variety of surgery variables which describe the performed anterior cruciate ligament (ACL) reconstruction and the interaction among them in the definition of postoperative outcome. Seventeen patients who underwent navigated Single Bundle plus Lateral Plasty ACL reconstruction were enrolled in the study. Static laxity was evaluated as the value of anterior/posterior displacement at 30° and at 90° of flexion, internal/external rotation at 30° and 90° of knee flexion, varus/valgus test at 0° and 30° of flexion. The evaluated surgical variables were analyzed through a multivariate analysis defining the following models: AP30_estimate, AP90_estimate, IE30_estimate, IE90_estimate, VV0_estimate, VV30_estimate. Surgical variables has been defined as the angles between the tibial tunnel and the three planes, the lengths of the tunnel and the relationship between native footprints and tunnels. An analogous characterization was performed for the femoral side. Performance and significance of the defined models have been quantified by the correlation ratio (η²) and the corresponding p-value (*p < 0.050). The analyzed models resulted to be statistically significant (p < 0.05) for prediction of postoperative static laxity values. The only exception was the AP90_estimate model. The η² ranged from 0.568 (IE90_estimate) to 0.995 (IE30_estimate). The orientation of the tibial tunnel resulted to be the most important surgical variable for the performed laxity estimation. Mathematical models for postoperative knee laxity is a useful tool to evaluate the effects of different surgical variables on the postoperative outcome.     

Brazilian Pediatric Reference Data for Quantitative Ultrasound of Phalanges According to Gender,Age, Height and Weight

Aims

To establish normative data for phalangeal quantitative ultrasound (QUS) measures in Brazilian students.

Methods

The sample was composed of 6870 students (3688 females and 3182 males), aged 6 to 17 years. The bone status parameter, Amplitude Dependent Speed of Sound (AD-SoS) was assessed by QUS of the phalanges using DBM Sonic BP (IGEA, Carpi, Italy) equipment. Skin color was obtained by self-evaluation. The LMS method was used to derive smoothed percentiles reference charts for AD-SoS according to sex, age, height and weight and to generate the L, M, and S parameters.

Results

Girls showed higher AD-SoS values than boys in the age groups 7–16 (p<0.001). There were no differences on AD-SoS Z-scores according to skin color. In both sexes, the obese group showed lower values of AD-SoS Z-scores compared with subjects classified as thin or normal weight. Age (r² = 0.48) and height (r² = 0.35) were independent predictors of AD-SoS in females and males, respectively.

Conclusion

AD-SoS values in Brazilian children and adolescents were influenced by sex, age and weight status, but not by skin color. Our normative data could be used for monitoring AD-SoS in children or adolescents aged 6–17 years.