To determine the role of telomere-mediated gene stability in hepatocarcinogenesis, we examined the telomere length of human liver with or without chronic liver diseases and hepatocellular carcinomas (HCC). The mean telomere restriction fragment (TRF) length of normal liver (n = 13), chronic hepatitis (n = 11), liver cirrhosis (n = 24) and HCC (n = 24) was 7.8 +/- 0.2, 7.1 +/- 0.3, 6.4 +/- 0.2 and 5.2 +/- 0.2 kb, respectively (mean +/- standard error). TRF length decreased with a progression of chronic liver diseases and that in HCC was significantly shorter than that in other chronic liver diseases (p < 0.05). The ratios of TRF length of HCC to that of corresponding surrounding liver of well differentiated (n = 7), moderately differentiated (n = 10) and poorly differentiated (n = 4) HCCs were 0.83 +/- 0.06, 0.75 +/- 0.05 and 0.98 +/- 0.09, respectively. The ratio of poorly differentiated HCC was significantly higher than that of moderately differentiated HCC (p < 0.05). A comparison between the size and telomere length ratio of moderately differentiated HCCs revealed a decrease of the ratio with size until it reached 50 mm in diameter. In contrast, the ratio increased as the size enlarged over 50 mm. These findings suggest that the gene stability of the liver cells mediated by the telomere is reduced as chronic liver disease progresses and that telomerase is activated in poorly differentiated HCC and moderately differentiated HCC over 50 mm in diameter.

A tocotrienol-rich fraction (TRF(25)) and novel tocotrienols (d-P(21)-T3 and d-P(25)-T3) of rice bran significantly lowered serum and low density lipoprotein cholesterol levels in chickens. The present study evaluated the effects of novel tocotrienols on lipid metabolism in swine expressing hereditary hypercholesterolemia. Fifteen 4-mo-old genetically hypercholesterolemic swine were divided into five groups (n = 3). Four groups were fed a corn-soybean control diet, supplemented with 50 microg of either TRF(25), gamma-tocotrienol, d-P(21)-T3 or d-P(25)-T3 per g for 6 wk. Group 5 was fed the control diet for 6 wk and served as a control. After 6 wk, serum total cholesterol was reduced 32-38%, low density lipoprotein cholesterol was reduced 35-43%, apolipoprotein B was reduced 20-28%, platelet factor 4 was reduced 12-24%, thromboxane B(2) was reduced 11-18%, glucose was reduced 22-25% (P<0.01), triglycerides were reduced 15-19% and glucagon was reduced 11-17% (P<0.05) in the treatment groups relative to the control. Insulin was 100% greater (P<0.01) in the treatment groups than in the control group. Preliminary data (n = 1) indicated that hepatic activity of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase was lower in the treatment groups, and cholesterol 7alpha-hydroxylase activity was unaffected. Cholesterol and fatty acid levels in various tissues were lower in the treatment groups than in control. After being fed the tocotrienol-supplemented diets, two swine in each group were transferred to the control diet for 10 wk. The lower concentrations of serum lipids in these four treatment groups persisted for 10 wk. This persistent effect may have resulted from the high tocotrienol levels in blood of the treatment groups, suggesting that the conversion of tocotrienols to tocopherols may not be as rapid as was reported in chickens and humans.

To define the complete sRNA population of the halophilic archaeon Haloferax volcanii, we employed high throughput sequencing. cDNAs were generated from RNA ranging in size from 17 to 500 nucleotides isolated from cells grown at three different conditions to exponential and stationary phase, respectively. Altogether, 145 intergenic and 45 antisense sRNAs were identified. Comparison of the expression profile showed different numbers of reads at the six different conditions for the majority of sRNAs. A striking difference in the number of sRNA reads was observed between cells grown under standard vs. low salt conditions. Furthermore, the six highest numbers of reads were found for low salt conditions. In contrast, only slight differences between sRNA reads at different growth temperatures were detected. Attempts to delete four sRNA genes revealed that one sRNA gene is essential. The three viable sRNA gene deletion mutants possessed distinct phenotypes. According to microarray analyses, the removal of the sRNA gene resulted in a profound change of the transcriptome when compared with the wild type. High throughput sequencing also showed the presence of high concentrations of tRNA derived fragments in H. volcanii. These tRF molecules were shown to have different amounts of reads at the six conditions analyzed. Northern analysis was used to confirm the presence of the tRNA-derived fragments.

This unit describes techniques to analyze telomeric length and telomerase activity in human cells. Telomere length can be determined by a modification of Southern blotting in which the analysis of chromosome terminal restriction fragments (TRFs) provides the average lengths of all telomeres in a cell population. Telomerase activity can be measured in vitro by a sensitive and efficient polymerase chain reaction (PCR)-based detection method, also known as telomeric repeat amplification protocol (TRAP). These assays can be used to study the in vitro cellular effects of aging and cancer treatments on telomere biology and telomerase activity.

The discovery of microRNA (miRNA) sorting into extracellular vesicles (EVs) revealed a novel mode of intercellular communication and uncovered a link between cellular endomembrane compartments and small RNAs in EV-secreting cells. Using a two-step ultracentrifugation procedure to isolate EVs released by T cells, we found that 45% of tRNA fragments (tRFs), but fewer than 1% of miRNAs, were significantly enriched in EVs compared with the corresponding cellular RNA. T cell activation induced the EV-mediated release of a specific set of tRFs derived from the 5' end and 3'-internal region of tRNAs without variable loops. Inhibition of EV biogenesis pathways specifically led to the accumulation of these activation-induced EV-enriched tRFs within multivesicular bodies (MVBs). Introducing antisense oligonucleotides to inhibit these tRFs enhanced T cell activation. Taken together, these results demonstrate that T cells selectively release tRFs into EVs via MVBs and suggest that this process may remove tRFs that repress immune activation.

This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G(0)/G(1) phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G(0)/G(1) phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.

Examined the role of the working alliance in the treatment of delinquent boys in community-based residential programs, clarifying the relation between therapeutic process and behavioral change. Horvath and Greenberg's (1989) Working Alliance Inventory was used to assess the therapeutic alliance between youth and staff after 3 weeks in treatment and again after 3 months. Achenbach's (1991) Child Behavior Checklist (CBCL; Youth Self-Report [YSR] and Teacher Report Form [TRF]) and recidivism scores were used to assess treatment progress and outcome. Results indicated that a positive working alliance assessed after 3 months in treatment related to positive psychological changes and predicted lower rates of recidivism. Unexpectedly, a positive working alliance assessed early in treatment was associated with negative outcomes (increased internalizing and externalizing symptoms and higher rates of recidivism). This finding suggests that for some delinquent youth initially optimistic assessments may be prognostic of slow progress or treatment failure.

The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

Human Neural Stem Cells (hNSCs) are excellent candidates for in vitro and in vivo molecular, cellular, and developmental research, and also for ex-vivo gene transfer and cell therapy in the nervous system. However, hNSCs are mortal somatic cells, and thus invariably enter an irreversible growth arrest after a finite number of cell divisions in culture. It has been proposed that this is due to telomere shortening. Here, we show that long-term cultured (up to 4 years) v-myc perpetuated hNSC lines do preserve short but stable and homogeneous telomeres (TRF and Q-FISH determinations). hNSC lines (but not strains) express high levels of telomerase activity, which is activated by v-myc, as demonstrated here. Telomerase activity is not constitutive, becoming non-detectable after differentiation (in parallel to v-myc down-regulation). hNSC lines also maintain a stable cell cycle length, mitotic potential, differentiation and neuron generation capacity, and do not express senescence-associated beta-galactosidase over years, as studied here. These data, collectively, help to explain the immortal nature of v-myc-perpetuated hNSC lines, and to establish them as excellent research tools for basic and applied neurobiological and translational studies.

Triple-negative breast cancer (TNBC) is a breast cancer subtype characterized by marked differences between White and Black/African-American women. We performed a systems-level analysis on datasets from The Cancer Genome Atlas to elucidate how the expression patterns of mRNAs are shaped by regulatory noncoding RNAs (ncRNA). Specifically, we studied isomiRs, that is, isoforms of miRNAs, and tRNA-derived fragments (tRF). In normal breast tissue, we observed a marked cohesiveness in both the ncRNA and mRNA layers and the associations between them. This cohesiveness was widely disrupted in TNBC. Many mRNAs become either differentially expressed or differentially wired between normal breast and TNBC in tandem with isomiR or tRF dysregulation. The affected pathways included energy metabolism, cell signaling, and immune responses. Within TNBC, the wiring of the affected pathways with isomiRs and tRFs differed in each race. Multiple isomiRs and tRFs arising from specific miRNA loci (e.g., miR-200c, miR-21, the miR-17/92 cluster, the miR-183/96/182 cluster) and from specific tRNA loci (e.g., the nuclear tRNAGly and tRNALeu, the mitochondrial tRNAVal and tRNAPro) were strongly associated with the observed race disparities in TNBC. We highlight the race-specific aspects of transcriptome wiring by discussing in detail the metastasis-related MAPK and the Wnt/β-catenin signaling pathways, two of the many key pathways that were found differentially wired. In conclusion, by employing a data- and knowledge-driven approach, we comprehensively analyzed the normal and cancer transcriptomes to uncover novel key contributors to the race-based disparities of TNBC.Significance: This big data-driven study comparing normal and cancer transcriptomes uncovers RNA expression differences between Caucasian and African-American patients with triple-negative breast cancer that might help explain disparities in incidence and aggressive character. Cancer Res; 78(5); 1140-54. ©2017 AACR.

Previous studies have revealed that tocotrienol-rich fractions (TRF) from palm oil inhibit the proliferation and the growth of solid tumors. The anticancer activity of TRF is said to be caused by several mechanisms, one of which is antiangiogenesis. In this study, we looked at the antiangiogenic effects of TRF. In vitro investigations of the antiangiogenic activities of TRF, delta-tocotrienol (deltaT3), and alpha-tocopherol (alphaToc) were carried out in human umbilical vein endothelial cells (HUVEC). TRF and deltaT3 significantly inhibited cell proliferation from 4 microg/ml onward (P < 0.05). Cell migration was inhibited the most by deltaT3 at 12 microg/ml. Anti-angiogenic properties of TRF were carried out further in vivo using the chick embryo chorioallantoic membrane (CAM) assay and BALB/c mice model. TRF at 200 microg/ml reduced the vascular network on CAM. TRF treatment of 1 mg/mouse significantly reduced 4T1 tumor volume in BALB/c mice. TRF significantly reduced serum vascular endothelial growth factor (VEGF) level in BALB/c mice. In conclusion, this study showed that palm tocotrienols exhibit anti-angiogenic properties that may assist in tumor regression.

Guillain-Barré syndrome (GBS) patients may worsen after initial treatment (treatment-related fluctuation [TRF]). It is difficult to distinguish GBS-TRF from chronic inflammatory demyelinating polyneuropathy with acute onset (A-CIDP). The authors compared 13 patients with A-CIDP with 11 patients with GBS-TRF and concluded that A-CIDP should be suspected when a patient with GBS deteriorates after 9 weeks from onset or when deterioration occurs three times or more. Maintenance treatment should then be considered.

Divalent metal-ion transporter-1 (DMT1) is required for iron uptake by the intestine and developing erythroid cells. DMT1 is also present in the liver, where it has been implicated in the uptake of transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. To test the hypothesis that DMT1 is required for hepatic iron uptake, we examined mice with the Dmt1 gene selectively inactivated in hepatocytes (Dmt1(liv/liv) ). We found that Dmt1(liv/liv) mice and controls (Dmt1(flox/flox) ) did not differ in terms of hepatic iron concentrations or other parameters of iron status. To determine whether hepatocyte DMT1 is required for hepatic iron accumulation, we crossed Dmt1(liv/liv) mice with Hfe(-) (/) (-) and hypotransferrinemic (Trf(hpx/hpx) ) mice that develop hepatic iron overload. Double-mutant Hfe(-) (/) (-) Dmt1(liv/liv) and Trf(hpx/hpx) ;Dmt1(liv/liv) mice were found to accumulate similar amounts of hepatic iron as did their respective controls. To directly assess the role of DMT1 in NTBI and TBI uptake, we injected (59) Fe-labeled ferric citrate (for NTBI) or (59) Fe-transferrin into plasma of Dmt1(liv/liv) and Dmt1(flox/flox) mice and measured uptake of (59) Fe by the liver. Dmt1(liv/liv) mice displayed no impairment of hepatic NTBI uptake, but TBI uptake was 40% lower. Hepatic levels of transferrin receptors 1 and 2 and ZRT/IRT-like protein 14, which may also participate in iron uptake, were unaffected in Dmt1(liv/liv) mice. Additionally, liver iron levels were unaffected in Dmt1(liv/liv) mice fed an iron-deficient diet.

CONCLUSIONS

Hepatocyte DMT1 is dispensable for hepatic iron accumulation and NTBI uptake. Although hepatocyte DMT1 is partially required for hepatic TBI uptake, hepatic iron levels were unaffected in Dmt1(liv/liv) mice, suggesting that this pathway is a minor contributor to the iron economy of the liver.

OBJECTIVE

The aim of this study was to determine whether telomere length is an independent prognostic factor for the prevention and survival of colorectal cancer.

METHODS

Terminal restriction fragment (TRF) length was determined by Southern blot in tumours and paired normal tissue samples from 147 patients with sporadic colorectal cancer who had undergone surgery. The TRF length ratio (TRFLR) was determined as the ratio between the length of the patient's tumour and normal tissue.The classification and regression tree technique was used to determine optimal cut-off values (≤ 1 or>> 1).

RESULTS

Mean TRF length was 6.79 Kbp (1.19-13.99) in tumour tissue and 7.81 Kbp (3.63-15.70) in normal mucosa (P < 0.001). Mean TRFLR was 0.88. Telomere length and telomere length ratio were not correlated with any clinicopathological factors. In univariate analysis, overall survival was related to N stage (lymph node +/-; P = 0.002), TNM classification (P = 0.019) and TRFLR (≤ 1 or>> 1; P = 0.014). In multivariate analysis, overall survival was significantly associated with TRFLR and N stage. Colorectal cancer patients with TRFLR ≤ 1 and negative lymph node involvement had a higher overall survival rate.

CONCLUSIONS

Telomere length ratio is an independent prognostic factor for survival in colorectal cancer patients, and the telomere lengths in the normal and tumour mucosa of the same patient present with parallel behaviour.

OBJECTIVE

To investigate relevant adults' perceptions of behavioral problems in 66 children with newly diagnosed "epilepsy only" and in 63 healthy gender-matched classmates.

METHODS

Parents' and teachers' perceptions of the children's behavioral problems were quantified by using the Total Problem score of the Child Behavior Checklist (CBCL) and the Teacher's Report Form (TRF), after correction for epilepsy-related item ambiguity. Questionnaires were filled out immediately after diagnosis and 3 and 12 months later. Relations with demographic and educational variables, school attitudes, and interview-derived prior adversities were analyzed.

RESULTS

As a group, children with cryptogenic rather than those with idiopathic epilepsy have more behavioral problems than do healthy classmates. Family troubles and long-standing behavioral and learning problems are associated with more behavioral problems. The child's adaptation to the adversity of epilepsy onset is important. No adverse effect of antiepileptic drug (AED) treatment was found. Although the percentages of patients with clinically relevant (mean of controls + 2 SD) behavioral problems are consistently 25% (parents) and 22% (teachers), at each assessment, different children contribute to these percentages. In not a single child did parents and teachers agree on the presence of clinically relevant behavioral problems.

CONCLUSIONS

(a) Behavioral problems are common in "epilepsy only," but are not persistent. (b) Agreement between parent's and teacher's perceptions of behavior is low. (c) Behavioral problems are perceived to occur already in the earliest stage of the disease.

The vitamin E component of palm oil provides a rich source of tocotrienols which have been shown previously to be growth inhibitory to two human breast cancer cell lines: responsive MCF7 cells and unresponsive MDA-MB-231 cells. Data presented here shows that the tocotrienol-rich fraction (TRF) of palm oil and individual fractions (alpha, gamma and delta) can also inhibit the growth of another responsive human breast cancer cell line, ZR-75-1. At low concentrations in the absence of oestrogen tocotrienols stimulated growth of the ZR-75-1 cells, but at higher concentrations in the presence as well as in the absence of oestradiol, tocotrienols inhibited cell growth strongly. As for MCF7 cells, alpha-tocopherol had no effect on growth of the ZR-75-1 cells in either the absence or presence of oestradiol. In studying the effects of tocotrienols in combination with antioestrogens, it was found that TRF could further inhibit growth of ZR-75-1 cells in the presence of tamoxifen (10(-7) M and 10(-8) M). Individual tocotrienol fractions (alpha, gamma, delta) could inhibit growth of ZR-75-1 cells in the presence of 10(-8) M oestradiol and 10(-8) M pure antioestrogen ICI 164,384. The immature mouse uterine weight bioassay confirmed that TRF could not exert oestrogen antagonist action in vivo. These results provide evidence of wider growth-inhibitory effects of tocotrienols beyond MCF7 and MDA-MB-231 cells, and with an oestrogen-independent mechanism of action, suggest a possible clinical advantage in combining administration of tocotrienols with antioestrogen therapy.

BACKGROUND

Der p 2 is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variations on T cell and antibody responses has not been compared.

OBJECTIVE

To compare IgE antibody binding and T cell proliferation and cytokine release induced by variants of Der p 2.

METHODS

Peripheral blood mononuclear cells (PBMC) from 19 allergic and 15 non-allergic people were stimulated with recombinant variants of Der p 2. IL-5, IL-10, IL-13 and IFN-gamma were measured by a time resolved fluorescence (TRF) assay. Serum IgE antibody was measured using a solid-phase TRF assay.

RESULTS

Overall the most prevalent variant of Der p 2 (Der p 2. 0101) was the highest or approximately equal highest inducer of T cell proliferation and IL-5, IL-10, IL-13 and IFN-gamma release. The most divergent variant 0104 induced the next highest responses. The variants 0107 and 0108 showed interesting changes especially when the allergic status was considered. Responses to 0107 showed poor Th1/Th2 polarization and, except for IL-10 release, cytokine responses to 0108 were low for non-allergic subjects. The variant 0101 showed similar monoclonal antibody binding but moderately less IgE binding than the other variants.

CONCLUSIONS

The most prevalent variant, Der p 2. 0101, was the most active for T cell stimulation and although its IgE binding was slightly less than other variants that was highly correlated. The variant Der p 2. 0104 which contains the known common polymorphic changes had a response which was similar to Der p 2. 0101 and thus these two variants were the most stimulatory representations of Der p 2. The T cell responses to the less common variants 0107 and 0108 however, showed consistent differences demonstrating that changes in the sequence could change the cytokine response.

Neurotensin (NT), substance P (SP) and morphine sulfate (MS) elevate plasma prolactin and growth hormone levels in both normal or estrogen-progesterone pretreated male rats. By contrast, steroid priming is required for TRF to exhibit PRL-releasing activity. Naloxone, an opiate receptor blocker, reverses the stimulatory effect of MS only. Diphenhydramine, a histamine antagonist, inhibits the response to NT, SP and MS without affecting the response to TRF. These results suggest the involvement of a histaminic step in the action of NT, SP and MS. TRF, NT and SP do not appear to stimulate PRL and GH through activation of an opiate receptor.

OBJECTIVE

To explore the prevalence and progression of posttraumatic symptomatology (PTS), using emotional and behavioral indices of psychopathology in school-age children in the pathway of Hurricane Andrew (HI-IMPACT) and in a comparison group north of Miami (LO-IMPACT).

METHODS

Pynoos' Posttraumatic Stress Disorder Reaction Index and Achenbach's Teacher's Report Form (TRF) were administered 8 weeks and 32 weeks after the hurricane. In addition, 21 measures of disruptive behavior cataloged by Dade County Public Schools were aggregated and compared by grading period between pre- and posthurricane school years.

RESULTS

There were no statistically significant differences between the two schools in PTS at 8 weeks after the hurricane, although the children in the HI-IMPACT school were more likely to have severe PTS. TRF findings at 8 weeks revealed that children in the HI-IMPACT school evidenced lower means on the eight TRF scales and on the broader Internalizing and Externalizing measures. Analysis of the disruptive behavior revealed a drop in the marking period immediately after the hurricane in the HI-IMPACT area, but an opposite effect was observed in the LO-IMPACT area.

CONCLUSIONS

After the hurricane there was an initial increase in PTS and a concomitant decrease in other measures of behavior and psychopathology. PTS remained relatively high throughout the school year, but there was a rebound and subsequent normalization of the measures of disruptive behavior.

The telomere is composed of telomeric DNA and telomere-associated proteins. Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered. In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1. Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres. In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins. Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein. Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution.

OBJECTIVE

To examine the effects of different forms of family violence at two developmental stages by assessing a sample of 110 Israeli children, drawn from the case files of Israeli family service agencies, studied longitudinally in both middle childhood and adolescence.

METHODS

Information about the children's adjustment was obtained from parents, teachers, and the children themselves when the children averaged 10.6 and 15.9 years of age using the Child Behavior Checklist (CBCL), Teacher Report Form (TRF), Youth Self-Report (YSR), and Children's Depression Inventory (CDI). Information about the history of family violence was obtained from the mothers, fathers, children, and social workers.

RESULTS

The results paint a mixed picture of the effects of family violence on children and adolescents. The relationship between concurrent behavior problems and abuse group varied by informant and study phase, although they were strongest when children were the informants. Predictions regarding the relationship between early abuse and later adjustment were only partially confirmed. Different informants did not agree about which groups of children were most adversely affected, there was little stability over time in the pattern of reported effects, and children were more likely than other informants to report levels of maladjustment that varied depending on recent or concurrent exposure to family violence. Many families changed their abuse status over time, and children who were new victims at follow-up had the most internalizing problems. Girls were found to be at more risk for internalizing and externalizing behavior problems than boys.

CONCLUSIONS

Multiple informants are necessary to evaluate and assess the effects of family violence on children's behavior. Younger children may be more susceptible to the effects of family violence than older children, but problems manifest by some children may not carry over to adolescence. Changes in family and parenting practices, as well as in children's capacity to appraise and cope with family violence may help mitigate the adverse effects of family violence.

The Achenbach System of Empirically Based Assessment (ASEBA) for school-age children includes three instruments for assessing emotional and/or behavioral problems: Child Behavior Checklist (CBCL), completed by parents, Youth Self-Report (YSR), completed by adolescents and Teacher's Report Form (TRF), completed by teachers. This review article gives detailed information on the development of these forms in the United States and Brazil, describing the main changes to the items, scales and score cut-off points in original versions between 1991 and 2001, as well as the process involved in the translation, back-translation and cultural adaptation of the original questionnaires to develop the current official Brazilian versions of the CBCL, YSR and TRF. The utility of these tools for research and clinical practice is highlighted, mentioning epidemiological studies and evaluation of interventions conducted in Brazil. Researchers' and clinicians' doubts regarding the correct use of the current official Brazilian versions are answered, giving examples of frequently asked questions relevant to the Brazilian context.

The present work aimed at the development and application of transferrin receptor (TrfR)-targeted sterically stabilized liposomes encapsulating anti-BCR-ABL siRNA or asODN. Transferrin was coupled to the surface of liposomes encapsulating siRNA or asODN through the postinsertion method. Cell association and internalization were assessed by flow cytometry and confocal microscopy, respectively. BCR-ABL mRNA and Bcr-Abl protein levels were evaluated by qRT-PCR and Western blot, respectively. Cell viability was assessed using the resazurin reduction method. The amount of coupled transferrin and the size and stability over time of the liposomes were very satisfactory and reproducible. The siRNA encapsulation yield was dependent on the concentration of the encapsulation buffer used (20 or 300 mM), as opposed to asODN encapsulation yield which was high for both concentrations tested. Cell association and internalization studies were performed in leukemia cell lines treated with liposomes coupled to Trf (Trf-liposomes) or albumin (BSA-liposomes) or with nontargeted liposomes (NT-liposomes) encapsulating fluorescently labeled siRNA (Cy3-siRNA). These experiments clearly indicated that BSA- and NT-liposomes have no ability to promote the delivery of the encapsulated nucleic acids and that the Trf-liposomes deliver the nucleic acids by a Trf receptor-dependent mechanism. The Trf-liposomes encapsulating siRNA or asODN promote sequence-specific down-regulation of the BCR-ABL mRNA, although a certain extent of nonspecific sequence effects at the protein and cell viability level were observed. Overall, our results indicate that Trf-liposomes encapsulating gene silencing tools allow combining molecular and cellular targeting, which is a valuable approach for cancer treatment.

Transcriptional regulation by transcriptional regulatory factors (TRFs) of their target TRF genes is central to the control of gene expression. To study a static multi-tiered inter-TRF regulatory network in the human hepatoma cells, we have applied a Matrix RNAi approach in which siRNA knockdown and quantitative RT-PCR are used in combination on the same set of TRFs to determine their interdependencies. This approach focusing on several liver-enriched TRF families, each of which consists of structurally homologous members, revealed many significant regulatory relationships. These include the cross-talks between hepatocyte nuclear factors (HNFs) and the other TRF groups such as CCAAT/enhancer-binding proteins (CEBPs), retinoic acid receptors (RARs), retinoid receptors (RXRs) and RAR-related orphan receptors (RORs), which play key regulatory functions in human hepatocytes and liver. In addition, various multi-component regulatory motifs, which make up the complex inter-TRF regulatory network, were identified. A large part of the regulatory edges identified by the Matrix RNAi approach could be confirmed by chromatin immunoprecipitation. The resultant significant edges enabled us to depict the inter-TRF TRN forming an apparent regulatory hierarchy of (FOXA1, RXRA) ->> TCF1 ->> (HNF4A, ONECUT1) ->> (RORC, CEBPA) as the main streamline.