Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Keywords: KRAS; RAS; SAFB; farnesyltransferase; prenyltransferase.

BACKGROUND

We hypothesized that the effect of pimobendan (UD-CG 115 BS) to increase calcium sensitivity of contractile protein might result in less myocardial oxygen consumption (VO2) in comparison with dobutamine when they enhance ventricular contractility to the same extent. To examine this hypothesis, we compared the effects of pimobendan and dobutamine on left ventricular contractility and energetics using the frameworks of Emax (contractility index) and the relation between VO2 and PVA (systolic pressure-volume area, a measure of left ventricular total mechanical energy).

RESULTS

We measured VO2, Emax, PVA, and force-time integral (FTI) in excised, cross-circulated, nonfailing dog hearts. The slope of the VO2-PVA relation reciprocally indicates the efficiency from PVA-dependent VO2 to the total mechanical energy (contractile efficiency). The VO2 intercept of the VO2-PVA relation, i.e., PVA-independent VO2, reflects energy utilization for excitation-contraction coupling. The ratio of FTI to PVA-dependent VO2 can be called contractile economy. Both drugs comparably enhanced Emax. Although the contractile economy was greater by 14 +/- 19% (p less than 0.05) for pimobendan than for dobutamine, the contractile efficiency was similar between the two drugs. Oxygen cost of contractility, defined as the slope of the relation between the PVA-independent VO2 and Emax, was the same between the two drugs. Other mechanoenergetic effects of both drugs were similar except for a greater coronary vasodilating effect of pimobendan.

CONCLUSIONS

Pimobendan has almost the same mechanoenergetic effects as dobutamine but slightly greater contractile economy and coronary vasodilation. The calcium-sensitizing effect of pimobendan did not save the oxygen cost of contractility.

This study assessed the effect of different semen storage temperatures and the influence of semen pooling in semen viability. In experiment 1, semen samples (n = 30) of five Majorera bucks were individually processed [Individual semen (IS)] and after the first dilution (Tris-yolk extender), semen-diluted aliquots from each male were pooled semen (PS). Thereafter, semen samples (IS and PS) were preserved as fresh semen (37 and 20°C), chilled semen (4°C) and frozen semen. Sperm motility and the percentage of abnormal sperm cells and intact membrane acrosomes were defined. Semen preservation at 20 and 4°C did not modify the quality of spermatozoa for the first 24 h, but the conservation at 37°C caused a dramatic fall in the semen motility from 12 h onwards. Furthermore, the longevity of frozen-thawed semen was limited to 4-6 h. No differences were observed in semen parameters when PS was compared with semen from individual males in any of the preservation protocols assessed. In experiment 2, 120 goats were distributed in four experimental groups: in group fresh individual semen (FIS, n = 30) and group frozen-thawed individual semen (FTIS, n = 30), does were transcervically inseminated with fresh semen and frozen-thawed semen from each individual male, respectively, and in group fresh pooled semen (FPS, n = 30) and group frozen-thawed pooled semen (FTPS, n = 30), goats were transcervically inseminated with FPS and FTPS, respectively. The kidding rate was very close in the FIS and FPS groups (70.0% and 73.7%, respectively), and no significant differences were observed in the fertility rate between FTIS and FTPS. The results of this study confirmed that semen samples may be preserved satisfactorily for 24 h both at 20 and 4°C. In addition, the mixture of semen of different bucks did not significantly modify the semen parameters when compared with semen from individual males.

OBJECTIVE

Toll-like receptor 4 (TLR4) promotes inflammation in hemodialysis patients (HD). A soluble form of extracellular TLR4 (sTLR4) has been recently characterized, which showed the ability to attenuate TLR4 signalling. In this study, we describe the sTLR4 profile in regular HD patients.

METHODS

In a cross-sectional study we enrolled forty prevalent HD patients (68.2 ± 16.3 years, twenty-five males) with a median dialysis vintage of 41 months. Nineteen patients were undergoing standard bicarbonate HD (BHD) and 21 patients on-line hemodiafiltration (HDF). Ten healthy sex-matched subjects constituted the controls (C).

METHODS

Before and after the HD session, serum was tested for sTLR4 levels by ELISA. Moreover, clinical and biochemical data were collected, including body mass index, albumin, and C-reactive protein (CRP) levels. Body composition was expressed as a 3-compartment model, providing lean tissue index and fat tissue index (FTI).

METHODS

Describe the profile of sTLR4 in HD patients, evaluating the correlations among sTLR4 levels and the main clinical characteristics, inflammatory and nutritional parameters.

RESULTS

Patients with subclinical inflammation (i.e., high CRP levels without clinical symptomatology) presented higher sTLR4 levels (0.42 ± 0.25 ng/mL) with respect to both C and not inflamed HD patients (0.23 ± 0.19 ng/mL, P < .05). There was a significant direct correlation between predialysis sTLR4 and body mass index, FTI (r = 0.55), and CRP levels (r = 0.52) and inverse correlation with lean tissue index and albumin (r = -0.4). In multivariate analysis, sTLR4 resulted directly associated with FTI (P = .038). Notably, sTLR4 levels resulted higher in bicarbonate hemodialysis versus hemodiafiltration (0.37 ± 0.18 vs. 0.19 ± 0.21 ng/mL, P < .05).

CONCLUSIONS

sTLR4 correlates with inflammatory and nutritional parameters, presenting as a new potential player in modulating subclinical inflammation in HD patients.

Experiments designed to determine the accuracy of the Corning Immophase Free T4 assay revealed that there was a marked perturbation of thyroxine binding to the serum proteins during the assay; binding of 125I-T4 to the immobilised T4 antibody in the absence of merthiolate increased considerably with buffer dilution of serum; and the free thyroxine concentration declined significantly during pregnancy. These changes in measured fT4 were not observed with an equilibrium dialysis-radioimmunoassay procedure. The assay was precise, easily performed, and as effective as the free thyroxine index (FTI) in diagnosing thyroid disease. We conclude that the assay does not provide an accurate quantitative estimation of serum fT4 concentration in samples with elevated TBG concentration and that the kinetic principles on which the assay is based are altered as serum binding-protein concentration is reduced (serum dilution). Both FTI and fT4 (Corning) data in pregnant patients should be interpreted with caution and with reference to clinical symptomatology and other thyroid function tests.

The fatal toxicity index (FTI) is the absolute number of fatal poisonings caused by a particular drug divided by its consumption figure. Consequently, it is a useful measure in evaluating toxicity of the drug and its relevance in fatal poisonings. In this study, we assessed the FTI of medicinal drugs in 3 years (2005, 2009, and 2013) in Finland. As the measure of drug consumption, we used the number of defined daily doses (DDD) per population in each year. There were 70 medicinal drugs in Finland for which the mean FTI expressed as the number of deaths per million DDD over the three study years was higher or equal to 0.1. The Anatomical Therapeutic Chemical (ATC) classification system was used for the classification of the active ingredients of medicinal drugs according to the organ or system which they act on. Of these 70 drugs, 55 drugs (78.6 %) acted on the nervous system (denoted by ATC code N), 11 (15.7 %) on the cardiovascular system (C), three (4.3 %) on the alimentary tract and metabolism (A), and one (1.4 %) on the musculoskeletal system (M). The nervous system drugs consisted of 20 psycholeptics, (ATC code N05), 20 psychoanaleptics (N06), eight analgesics (N02), six antiepileptics (N03), and one other nervous system drug (N07). The highest individual FTIs were associated with the opioids methadone, dextropropoxyphene, oxycodone, tramadol, and morphine; the antipsychotics levomepromazine and chlorprothixene; and the antidepressants doxepin, amitriptyline, trimipramine, and bupropion. Buprenorphine was not included in the study, because most of the fatal buprenorphine poisonings were due to smuggled tablets. A clearly increasing trend in FTI was observed with pregabalin and possibly with bupropion, both drugs emerging as abused substances.

Objective: To evaluate the diagnostic value of FDG PET-CT metabolic parameters and Deauville-like 5 point-scale to predict malignancy in a population of patients presenting focal thyroid incidentaloma (fTI). Design: This retrospective study included 41 fTI, classified according to cytological and histological data as benign (BL) or malignant lesion (ML). FDG PET-CT semi-quantitative parameters (SUVmax, SUVmean, SUVpeak, MTV, TLG), tumor to liver SUVmean ratio (TLRmax and TLRmean), tumor to blood-pool SUVmean ratio (TBRmax and TBRmean) were calculated. Each fTI was also classified on a Deauville-like 5-point scale (DS) currently used in lymphoma. Comparison between BL and ML was performed for each parameter and a ROC analysis was conducted. Results: All quantitative PET metabolic parameters (SUV parameters, volume based parameters and SUV ratio) were higher in ML compared with BL, yet no significant difference was reported. fTI (uptake) malignancy rate according to DS grades 2, 3, 4, and 5 was, respectively, 25% (1 of 4), 28.6% (2 of 7), 8.3% (1 of 12), and 33.3% (6 of 18) with no significant difference between ML and BL groups. Results of ROC analysis showed that mean TBR had the highest AUC in our cohort (0.66 95%CI [0.41; 0.91]) with a cut-off value of 2.2. Specificity of MTV and TLG was 100% (cut-off values: MTV 9.6 ml, TLG 22.9 g) and their sensitivity was 30 and 40%, respectively. Conclusion: Our study did not highlight any FDG PET/CT parameter predictor of fTI malignancy.

In the incessant search for innovative cancer control strategies, this study was devoted to the design, synthesis and pharmacological evaluation of dual inhibitors of farnesyltransferase and tubulin polymerization (FTI/MTIs). A series of indolizine-phenothiazine hybrids 16 (amides) and 17 (ketones) has been obtained in a 4-step procedure. The combination of the two heterocycles provided potent tubulin polymerization inhibitors with similar efficiency as the reference phenstatin and (-)-desoxypodophyllotoxin. Ketones 17 were also able to inhibit human farnesyltransferase (FTase) in vitro. Interestingly, three molecules 17c, 17d and 17f were very effective against both considered biological targets. Next, nine indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b were evaluated for their cell growth inhibition potential on the NCI-60 cancer cell lines panel. Ketones 17a-f were the most active and displayed promising cellular activities. Not only they arrested the cell growth of almost all tested cancer cells, but they displayed cytotoxicity potential with GI₅₀ values in the low nanomolar range. The most sensitive cell lines upon treatment with indolizine-phenothiazine hybrids were NCI-H522 (lung cancer), COLO-205 and HT29 (colon cancer), SF-539 (human glioblastoma), OVCAR-3 (ovarian cancer), A498 (renal cancer) and especially MDA-MB-435 (melanoma). Demonstrating the preclinical effectiveness of these dual inhibitors can be crucial. A single dual molecule could induce a synergy of antitumor activity, while increasing the effectiveness and reducing the toxicity of the classical combo treatments currently used in chemotherapy.

Keywords: Antitumor agent; Dual inhibitor; Farnesyltransferase inhibitor; Hybrids; Indolizine; Phenothiazine; Tubulin polymerization.

OBJECTIVE

This study was designed to assess the diagnostic potency of different androgens in hyperandrogenaemia criterion on polycystic ovary syndrome (PCOS) based on receiver operator characteristic (ROC) curves analysis.

METHODS

We evaluated 55 PCOS patients and 27 healthy fertile women (control). Androgen evaluation included bio-available testosterone (BAT) by ammonium sulphate precipitation, Free Testosterone Index (FTI), androstenedione (A), total testosterone and dehydroepiandrosterone sulphate (DHEA-S).

RESULTS

The androgen tests with the best diagnostic capacities were FTI and BAT. Although T and A had similar diagnostic potencies, A detected 5% of PCOS patients that could not be recognised by FTI, BAT (%), or T. The association of FTI, BAT (%) and A identified 96.36% of the hyperandrogenaemic patients. DHEA-S showed a wide dispersion of values and therefore poor discriminatory competence.

CONCLUSIONS

This study suggests that routine androgen evaluation in PCOS should include FTI, BAT and A to avoid misdiagnosis. ROC curve analysis of these tests on patients with the complete spectrum of PCOS phenotypes is needed to confirm these results.

A mathematical formula was derived from an active cross-bridge model to express the changes in the active myocardial force which occurred during systole. Using the formula and the assumption that the energy expenditure for cross-bridge cycling (Um) was a linear function of the force-time integral (FTI), we developed formulae describing the left ventricular Um versus FTI relation, the Um versus force relation, and the Um versus pressure-volume area (PVA) relation. There were strong disagreements between the model predictions and the experimental findings relating oxygen consumption of the heart versus the PVA relation. These differences may have resulted from the oversimplification of important mechanical and/or biochemical properties of the myocardium in the model. However, the model appeared to accurately reproduce the Fenn effect (effect of contraction modes on energy liberation) for the myocardium as well as the effect of catecholamine infusion, hypothermia, and hypothyroidism on the changes in the binding rate of Ca2+ with the regulatory proteins, the myosin ATPase activity, the peak force developed, and the myocardial energy expenditure. We present this work as an intermediate step towards a complete theoretical linkage between the molecular biology, dynamics, and energetics of the human heart.

Assessment of dental health in the primary dentition of preterm infants (PTI) including investigation of mother- and infant-related risk factors in a case-control study design.

One hundred twenty-eight infants aged 3-4 years were included. Sixty-four PTI (27 males) were randomly selected from the preterm registry of the Jena University Hospital. As a control group served 64 full-term infants (FTI) recruited from the Department of Paediatric Dentistry, matched for age and sex. Dental examinations were provided by one dentist under standard clinical conditions. Caries was scored using the International Caries Detection and Assessment System (ICDAS II) and the DMFT, gingival health using the Periodontal Screening Index, and developmental defects of enamel using the DDE index. Mother- and infant-related factors were collected via a questionnaire and from medical records.

The caries prevalence was 50.0% (ICDAS II >0) in PTI and 12.5% (ICDAS II >0) in FTI. The caries experience was higher in PTI (DMFT 1.0 ± 3.1) than in FTI (DMFT 0.3 ± 1.0). PTI had a higher risk of caries (OR 7.0), initial lesions (OR 6.2), DDE (OR 7.5), and gingivitis (OR 6.5) than FTI. The highest risk occurred in PTI with an extremely low birth weight (<1,000 g). A higher risk of DDE was present when mothers suffered from illness during pregnancy (OR 3.9). A higher risk of caries was revealed in infants with respiratory syndrome (OR 6.2) or when their mothers had a lower socioeconomic status (OR 6.3).

PTI had less healthy teeth than FTI and are at a higher risk for DDE, caries, and gingivitis. The poorer dental health in PTI is associated with a low birth weight, a low socioeconomic status, and mothers' illness during pregnancy.

This study investigated flow-through immunocapture (FTI), using the Pathatrix device, followed by plating on xylose lysine desoxycholate (XLD) agar (FTI-XLD) or analysis by real-time PCR (FTI-PCR) for the detection of Salmonella on smooth tomato surfaces and in potato salad and ground beef within 8 h. Food samples were inoculated with an appropriate dilution of a five-serovar Salmonella cocktail and enriched for 5 h. Following enrichment, samples were analyzed by the FTI-XLD and FTI-PCR methods. Food samples were also analyzed by a modified U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Salmonella culture method for comparison. Salmonella inoculated at 10(0) CFU per tomato or 10(0) CFU/25 g was detected by the FTI-XLD method in 6, 8, and 4 of 10 samples for tomatoes, potato salad, and ground beef, respectively. Salmonella inoculated at 10(0) CFU per tomato or 10(0) CFU/25 g was detected by the FTI-PCR method in 8, 9, and 9 of 10 samples for tomatoes, potato salad, and ground beef, respectively. The FTI-PCR method achieved significantly higher (P < 0.05) detection of Salmonella on tomatoes, whereas the FTI-XLD method achieved significantly lower (P < 0.05) detection of Salmonella in ground beef when compared with the modified BAM Salmonella culture method; however, all other comparisons to the modified BAM method were not significantly different. The FTI-XLD method demonstrated the ability to isolate presumptive Salmonella colonies up to 48 hfaster than did the modified BAM Salmonella culture method.

Recent work (Cembrowski et al, Am J Clin Pathol 1984;81:492-499) has shown that the average of selected patient data can theoretically be used to demonstrate systematic errors. We formerly demonstrate that the sensitivity or power of the 'average of patients' (AOP) is related to the index (NpSw2)/Sp2 where Np is the number of patient results averaged, Sw the within-run component of the standard deviation of the analytical method and Sp the standard deviation of the trimmed population. The utility of the AOP can therefore be assessed rapidly without power function curves or the calculation of the daily AOP itself. The index showed that, for 12 radioimmunoassays done in our laboratory, the power of the AOP was greatest for T3 uptake, then FTI, then T4. Daily AOP were followed for these tests with control results on parallel Shewhart plots. We also applied an exponential smoothing procedure on the AOP and control results to improve trend detection. These analyses demonstrated that the AOP can improve the detection of systematic errors and the differentiation of true errors from errors affecting only control specimens.

Rocker profiles are commonly used in the prevention of diabetic foot ulcers. Rockers are mostly stiffened to restrict toe plantarflexion to ensure proper offloading. It is also described that toe dorsiflexion should be restricted. However, the difference in effect on plantar pressure between rigid rockers that restrict this motion and flexible rockers that do not is unknown. In-shoe plantar pressure data were collected for a control shoe and the same shoe with rigid and flexible rockers with the apex positioned at 50% and 60%. For 29 healthy female adults peak plantar pressure (PP), maximum mean pressure (MMP) and force-time integral (FTI) were determined for seven regions of the foot. Generalized estimate equation was used to analyse the effect of the different shoes on the outcome measures for these regions. Compared to the control shoe a significant increase of PP and FTI was found at the first toe for both rigid rockers and the flexible rocker with the apex positioned at 60%, while MMP was significantly increased in rockers with an apex position of 60% (p<0.001). PP at the first toe was significantly lower in flexible rockers when compared to rigid rockers (p<0.001). For both central and lateral forefoot PP and MMP were significantly more reduced in rigid rockers (p<0.001), while for the medial forefoot no differences were found. The use of rigid rockers results in larger reductions of forefoot plantar pressures, but in worse increase of plantar pressures at the first toe compared to rockers that allow toe dorsiflexion.

The effect of isoflurane (Forane) anaesthesia and surgery on thyroid function was investigated in nine male patients. Isoflurane anaesthesia alone for 30 minutes prior to the start of surgery increased plasma triiodothyronine uptake (T3U) and thyroxine (T4) level by 18 and 20 per cent, respectively. Free thyroxine index (FTI) values in blood increased by 62 per cent during the same period. One hour of surgical procedure further elevated these parameters. Our data indicate that isoflurane anaesthesia and surgery increase circulating thyroxine in man.

Clinical and biochemical indices of thyroid function were compared in 18 euthyroid control subjects and 54 patients at risk of developing hypothyroidism, and the assessment repeated after a four-week trial of oral triiodothyronine (T3) 40 microgram daily. Patients with minor elevation of the pre-treatment serum TSH concentration (5-15 microU/ml) showed significant reduction in the free thyroxine index (FTI), prolongation of the basal ankle reflex half-relasation time (ART), and shortening of the ART after oral T3 treatment, when compared either to control subjects or to patients with a normal TSH level. It is concluded that patients with minimal TSH elevation have minor but parallel abnormalities of both thyroid hormone concentration and action.

In 35 patients with simple multinodular goiter and with normal preoperative TSH reserve, the mean basal TSH and the mean TSH response to TRH increased significantly 5-15 days and 1-15 months following partial thyroidectomy. The postoperative mean TSH values were also greater than the age-related normal mean values. The incidence of postoperative supranormal TSH values and the incidence of postoperative increase in basal and TRH-stimulated TSH did not differ in the early and late postoperative course. In the latter the FTI had decreased significantly in the euthyroid range as compared to the preoperative value, whereas the mean serum T3 had not increased significantly. The patients were divided into two subgroups according to the anatomo-pathological finding and ensuing type of goiter resection (either selective ablation of non-functioning parenchyma with normal remnants, or resection of functioning and non-functioning parenchyma in the presence of goitrous remnants); the postoperative increase in the mean basal and TRH-stimulated serum TSH values was significant only in the non-selective ablation subgroup. Preclinical hypothyroidism was noted post surgery in 6 patients with supranormal preoperative TSH reserve. The results demonstrate a decreased thyroid reserve, as reflected in a persistent increase in thyrotropic function following resection of simple multinodular goiter in patients from a mildly endemic area. Long-term follow-up and thyroid hormone substitution may chiefly be indicated following non-selective goiter resection, in order to eliminate or prevent preclinical hypothyroidism and to prevent development of recurrent goiter.

Quantitative structure activity relationship (QSAR) models can be used to predict the activity of new drug candidates in early stages of drug discovery. In the present study, the information of the ninety two 2,5-diaminobenzophenone-containing farnesyltranaferase inhibitors (FTIs) were taken from the literature. Subsequently, the structures of the molecules were optimized using Hyperchem software and molecular descriptors were obtained using Dragon software. The most suitable descriptors were selected using genetic algorithms-partial least squares and stepwise regression, where exhibited that the volume, shape and polarity of the FTIs are important for their activities. The two-dimensional QSAR models (2D-QSAR) were obtained using both linear methods (multiple linear regression) and non-linear methods (artificial neural networks and support vector machines). The proposed QSAR models were validated using internal validation method. The results showed that the proposed 2D-QSAR models were valid and they can be used for prediction of the activities of the 2,5-diaminobenzophenone-containing FTIs. In conclusion, the 2D-QSAR models (both linear and non-linear) showed good prediction capability and the non-linear models were exhibited more accuracy than the linear models.

In 430 patients with a variety of thyroid disorders, linear discriminant analysis was used to select laboratory test combinations giving optimum diagnostic efficiency in thyroid function studies. Unexpectedly, TSH was found valuable in the diagnosis of hyperthyroidism and T3 in hypothyroidism. These test combinations were found optimal for the separation of euthyroidism/hyperthyroidism: T3, TSH and T4; euthyroidism/hypothyroidism: Combined free thyroid hormone index (FTI) and TSH; and hyperthyroidism/euthyroidism/hypothyroidism: T3, TSH, T4, FTI and T3U. The latter test combination had a total efficiency of 94%. Cholesterol, achilles reflex time, PBI and radioiodine uptake measurement contributed little to the discrimination.