SINGLE INSTITUTION VARIABILITY IN INTENSITY MODULATED RADIATION TARGET DELINEATION FOR CANINE NASAL NEOPLASIA.

Contouring variability is a significant barrier to the accurate delivery and reporting of radiation therapy. The aim of this descriptive study was to determine the variation in contouring radiation targets and organs at risk by participants within our institution. Further, we also aimed to determine if all individuals contoured the same normal tissues. Two canine nasal tumor datasets were selected and contoured by two ACVR-certified radiation oncologists and two radiation oncology residents from the same institution. Eight structures were consistently contoured including the right and left eye, the right and left lens, brain, the gross tumor volume (GTV), clinical target volume (CTV), and planning target volume (PTV). Spinal cord, hard and soft palate, and bulla were contoured on 50% of datasets. Variation in contouring occurred in both targets and normal tissues at risk and was particularly significant for the GTV, CTV, and PTV. The mean metric score and dice similarity coefficient were below the threshold criteria in 37.5-50% and 12.5-50% of structures, respectively, quantitatively indicating contouring variation. This study refutes our hypothesis that minimal variation in target and normal tissue delineation occurs. The variation in contouring may contribute to different tumor response and toxicity for any given patient. Our results also highlight the difficulty associated with replication of published radiation protocols or treatments, as even with replete contouring description the outcome of treatment is still fundamentally influenced by the individual contouring the patient.

Preference for language of instruction of an English language learner with autism.

OBJECTIVE: To use a preference assessment to identify the preferred language of instruction (English or Spanish) for a child with autism living in a Spanish-speaking home and receiving school services in English only. METHODS: We used a concurrent chains method to evaluate the participant's preference for English or Spanish instruction. Colored microswitches represented English instruction, Spanish instruction and no instruction (control). Switch presses resulted in instruction and reinforcement in either English or Spanish, or no instruction and no reinforcement. After session 10, switches were reprogrammed to control for color bias. RESULTS: The participant chose Spanish instruction most frequently. CONCLUSIONS: The participant preferred to receive instruction in Spanish. These data support previous research indicating the importance of child preference when individualizing educational programs for children with autism and that language of instruction is an important variable when working with children with autism who experience multiple languages across home and school.

Increased Butyrate Production During Long-Term Fermentation of In Vitro-Digested High Amylose Cornstarch Residues with Human Feces.

An in vitro semi-continuous long-term (3 wk) anaerobic incubation system simulating lower gut fermentation was used to determine variability in gut microbial metabolism between 4 predigested high amylose-resistant starch residues (SR): SRV, SRVI, SRVII, and SRGEMS in human fecal samples. Subjects participated twice, 5 mo apart: 30 in Phase I (15 lean, 9 overweight and 6 obese), 29 in Phase II (15 lean, 9 overweight, 5 obese); 13 of 15 lean subjects participated in both phases. Of the 4 SRs, SRV displayed the highest gelatinization temperature, peak temperature, enthalpy changes, and the least digestibility compared with the other SRs. In both phases, compared with blank controls, all SRs increased butyrate ∼2-fold which stabilized at week 2 and only SRV caused greater propionate concentration (∼30%) after 3 wk which might have been partly mediated by its lesser digestibility. Fecal samples from lean and overweight/obese subjects incubated with SRs showed similar short-chain fatty acid production across both time points, which suggests that resistant starch may benefit individuals across BMIs.

Ethnicity reporting practices for empirical research in three autism-related journals.

This review examines ethnicity reporting in three autism-related journals (Autism, Focus on Autism and Other Developmental Disabilities, and Journal of Autism and Developmental Disorders) over a 6-year period. A comprehensive multistep search of articles is used to identify ethnicity as a demographic variable in these three journals. Articles that identified research participants' ethnicity were further analyzed to determine the impact of ethnicity as a demographic variable on findings of each study. The results indicate that ethnicity has not been adequately reported in these three autism related journals even though previous recommendations have been made to improve inadequacies of descriptive information of research participants in autism research (Kistner and Robbins in J Autism Dev Disord 16:77-82, 1986). Implications for the field of autism spectrum disorders are discussed in addition to further recommendations for future research.

Impact of the molecular weight, viscosity, and solubility of ß-glucan on in vitro oat starch digestibility.

The impacts of the molecular weight (MW), viscosity, and solubility of ß-glucan on the rate of in vitro starch digestion and estimated glycemic index (GI) were evaluated. Extracted oat starch and ß-glucan suspensions with high, medium, and low MW were heated to gelatinize the starch. The viscosity increased and the solubility decreased with an increase in the MW of ß-glucan. The in vitro starch hydrolysis of the mixtures and a control, white bread, increased as the digestion time increased. As the MW of ß-glucan increased, the starch hydrolysis decreased during in vitro digestion. The in vitro estimated GI of the mixture without ß-glucan, determined from the starch hydrolysis rate, was 88.3 for Jim and 80.0 for N979, which decreased to 68.4 and 66.8, respectively, with the inclusion of high-MW ß-glucan. The estimated GI values were negatively correlated with the ß-glucan peak and final viscosities (r = -0.81 and -0.82). These results illustrated the importance of viscosity attributed to the ß-glucan MW on starch hydrolysis during in vitro digestion. These findings will help to develop new food products with a low GI by using oat ß-glucan.

Genome-wide association study for oat (Avena sativa L.) beta-glucan concentration using germplasm of worldwide origin.

Detection of quantitative trait loci (QTL) controlling complex traits followed by selection has become a common approach for selection in crop plants. The QTL are most often identified by linkage mapping using experimental F(2), backcross, advanced inbred, or doubled haploid families. An alternative approach for QTL detection are genome-wide association studies (GWAS) that use pre-existing lines such as those found in breeding programs. We explored the implementation of GWAS in oat (Avena sativa L.) to identify QTL affecting ß-glucan concentration, a soluble dietary fiber with several human health benefits when consumed as a whole grain. A total of 431 lines of worldwide origin were tested over 2 years and genotyped using Diversity Array Technology (DArT) markers. A mixed model approach was used where both population structure fixed effects and pair-wise kinship random effects were included. Various mixed models that differed with respect to population structure and kinship were tested for their ability to control for false positives. As expected, given the level of population structure previously described in oat, population structure did not play a large role in controlling for false positives. Three independent markers were significantly associated with ß-glucan concentration. Significant marker sequences were compared with rice and one of the three showed sequence homology to genes localized on rice chromosome seven adjacent to the CslF gene family, known to have ß-glucan synthase function. Results indicate that GWAS in oat can be a successful option for QTL detection, more so with future development of higher-density markers.

In vitro digestion rate and estimated glycemic index of oat flours from typical and high ß-glucan oat lines.

The in vitro starch digestion rate and estimated glycemic index (GI) of oat flours and oat starches from typical and high ß-glucan oat lines were evaluated along with the impact of heating on starch digestion. Flour from oat lines ('Jim', 'Paul', IA95, and N979 containing 4.0, 5.3, 7.4, and 7.7% ß-glucan, respectively) was digested by pepsin and porcine pancreatin. To determine the impact of heating on starch digestion, oat slurries were prepared by mixing oat flour and water (1:8 ratio) and heating for 10 min prior to digestion. Viscosity, as measured on a Rapid Visco Analyzer, increased with increases in concentration and molecular weight of ß-glucan. The in vitro starch digestion of oat flours and a control, white bread made from wheat flour, increased as the digestion time increased. Starch digestion of oat flour was slower than that of the control (p < 0.05). Heat treatment of oat-flour slurries increased the starch digestion from a range of 31-39% to a range of 52-64% measured after 180 min of in vitro digestion. There were no differences in starch digestibility among oat starches extracted from the different oat lines. The GI, estimated by starch hydrolysis of oat flours, ranged from 61 to 67, which increased to a range of 77-86 after heating. Oat-flour slurries prepared from IA95 and N979 lines with high ß-glucan concentrations had lower GI values than did slurries made from Jim and Paul lines. Starch digestion was negatively correlated with ß-glucan concentrations in heated oat-flour slurries (R(2) = 0.92). These results illustrate that the oat soluble fiber, ß-glucan, slowed the rate of starch digestion. This finding will help to develop new food products with low GI by using oat ß-glucan.

Interactional effects of ß-glucan, starch, and protein in heated oat slurries on viscosity and in vitro bile acid binding.

Three major oat components, ß-glucan, starch, and protein, and their interactions were evaluated for the impact on viscosity of heated oat slurries and in vitro bile acid binding. Oat flour from the experimental oat line "N979" (7.45% ß-glucan) was mixed with water and heated to make oat slurry. Heated oat slurries were treated with α-amylase, lichenase, and/or proteinase to remove starch, ß-glucan, and/or protein. Oat slurries treated with lichenase or lichenase combined with α-amylase and/or proteinase reduced the molecular weight of ß-glucan. Heat and enzymatic treatment of oat slurries reduced the peak and final viscosities compared with the control. The control bound the least amount of bile acids (p < 0.05); heating of oat flour improved the binding. Heated oat slurries treated with lichenase or lichenase combined with α-amylase and/or proteinase bound the least amount of bile acid, indicating the contribution of ß-glucan to binding. Oat slurries treated with proteinase or proteinase and α-amylase together improved the bile acid binding, indicating the possible contribution of protein to binding. These results illustrate that ß-glucan was the major contributor to viscosity and in vitro bile acid binding in heated oat slurries; however, interactions with other components, such as protein and starch, indicate the importance of evaluating oat components as whole system.

Optimizing the molecular weight of oat ß-glucan for in vitro bile acid binding and fermentation.

A previous study showed ß-glucan with low molecular weight (MW, 1.56×10(5) g/mol) bound more bile acid and produced greater amounts of short-chain fatty acids (SCFA) than did ß-glucan with high MW (Mn=6.87×10(5) g/mol). In the current study, ß-glucan extracted from oat flour was fractionated into six different MW levels (high MW, 7.09×10(5); low level 1 (L1), 3.48×10(5); L2, 2.42×10(5); L3, 1.61×10(5); L4, 0.87×10(5); and L5, 0.46×10(5) g/mol) and evaluated to find the optimum MW affecting in vitro bile acid binding and fermentation. The ß-glucan fractions with 2.42×10(5)-1.61×10(5) g/mol (L2 and L3) bound the greatest amounts of bile acid. After 24 h of fermentation, no differences were found in total SCFA formation among L1, L2, L3, and L4 fractions; however, the high MW and L5 MW fractions produced lower amounts of total SCFA. Thus, the optimum MW of ß-glucan to affect both hypocholesterolemic and antitumorigenic in vitro effects was in the range of 2.42×10(5)-1.61×10(5) g/mol. This MW range also was the most water-soluble among the MWs evaluated.

Molecular weight and structure of water soluble (1→3), (1→4)-ß-glucans affect pasting properties of oat flours.

Seven experimental oat lines with high (5.9% to 7.2%), medium (5.3% to 5.5%), and low (4.4%) ß-glucan concentrations were evaluated for the effects of ß-glucan molecular weight (MW) and structure on viscosities of oat-flour slurries. The MW of ß-glucans was determined by size-exclusion high-performance liquid chromatography. The structural features of ß-glucans were measured by using fluorophore-assisted capillary-electrophoresis after complete hydrolysis with lichenase. The oat-slurry viscosities were measured on a Rapid Visco Analyser under 4 conditions: (1) without starch (amylolysis, removal of starch by α-amylase); (2) without ß-glucan (removal of ß-glucan by lichenase); (3) natural action of enzymes (autolysis, in sodium buffer); and (4) inhibition of enzymes (in silver nitrate solution). Excluding one line (regression outlier), significant correlations (P < 0.05) between peak MW of ß-glucan and viscosities of oat slurries were obtained under inhibition. The ratio of degree of polymerization (DP) 3/DP4 negatively correlated with viscosity under amylolysis, autolysis, and inhibition (P < 0.05). The amount of DP ≥ 5 negatively correlated with pasting final viscosity after ß-glucan removal by lichenase (P < 0.05). Positive correlations (P < 0.05) between the ratio of ß-(1→4)/ß-(1→3) linkages and viscosities under autolysis and inhibition were found. Overall, these findings demonstrated that the peak MW, ratio of DP3/DP4, amount of DP ≥ 5, and ratio of ß-(1→4)/ß-(1→3) linkages of ß-glucans impacted pasting properties of oat-flour slurries.

Patient factors that influence warfarin dose response.

Warfarin has long been the mainstay of oral anticoagulation therapy for the treatment and prevention of venous and arterial thrombosis. The narrow therapeutic index of warfarin, and the complex number of factors that influence international normalized ratio (INR) response, makes optimization of warfarin therapy challenging. Determination of the appropriate warfarin dose during initiation and maintenance therapy requires an understanding of patient factors that influence dose response: age, body weight, nutritional status, acute and chronic disease states, and changes in concomitant drug therapy and diet. This review will examine specific clinical factors that can affect the pharmacokinetics and pharmacodynamics of warfarin, as well as the role of pharmacogenetics in optimizing warfarin therapy.

Individual and interactional effects of ß-glucan, starch, and protein on pasting properties of oat flours.

Seven experimental oat lines with high (6.2-7.2%), medium (5.5-5.9%), and low (4.4-5.3%) ß-glucan concentrations were evaluated for contributions of ß-glucan, starch, protein, and their interactions, to pasting properties of oat flours by using a Rapid Visco Analyser (RVA, Newport Scientific, Warriewood, NSW, Australia). Significant correlations (P < 0.05) between ß-glucan concentration and pasting parameters of oat slurries were obtained under autolysis without 1 h of incubation, inhibition, and amylolysis. The relative decrease of viscosity after enzymatic hydrolysis of ß-glucan correlated with ß-glucan concentration (P < 0.05). These data demonstrated the important contribution of ß-glucan to pasting. The relative decrease of viscosity after either amylolysis or enzymatic removal of protein correlated with ß-glucan concentration (P < 0.1), which might be explained by the considerable contribution of the interaction of ß-glucan with starch and protein, to pasting. The viscosity decrease by hydrolysis of protein was much greater than the actual viscosity remaining after hydrolysis of both ß-glucan and starch, reconfirming the importance of interactions between protein and other oat components to pasting. Optimal multiple linear regression (MLR) models were generated to predict key pasting parameters in both buffer without 1 h of incubation and silver nitrate solution by using a stepwise procedure. The ß-glucan concentration alone or together with the concentration of starch, rather than protein, was selected as the predictor under certain conditions. These results illustrated the major unit contribution of ß-glucan, secondary unit contribution of starch, and minimal unit contribution of protein to pasting.

In vitro bile-acid binding and fermentation of high, medium, and low molecular weight beta-glucan.

The impact of beta-glucan molecular weight (MW) on in vitro bile-acid binding and in vitro fermentation with human fecal flora was evaluated. beta-Glucan extracted from oat line 'N979-5-4' was treated with lichenase (1,3-1,4-beta-D-glucanase) to yield high (6.87x10(5) g/mol), medium (3.71x10(5) g/mol), and low (1.56x10(5) g/mol) MW fractions. The low MW beta-glucan bound more bile acid than did the high MW beta-glucan (p<0.05). If the positive control, cholestyramine, was considered to bind bile acid at 100%, the relative bile-acid binding of the original oat flour and the extracted beta-glucan with high, medium, and low MW was 15, 27, 24, and 21%, respectively. Significant effects of high, medium, and low MW beta-glucans on total SCFA were observed compared to the blank without substrate (p<0.05). There were no differences in pH changes and total gas production among high, medium, and low MW beta-glucans, and lactulose. The low MW beta-glucan produced greater amounts of SCFA than the high MW after 24 h of fermentation. Among the major SCFA, more propionate was produced from all MW fractions of extracted beta-glucans than from lactulose. In vitro fermentation of extracted beta-glucan fractions with different MW lowered pH and produced SCFA, providing potential biological function.

In vitro fermentation of oat flours from typical and high beta-glucan oat lines.

Two publicly available oat (Avena sativa) lines, "Jim" and "Paul" (5.17 and 5.31% beta-glucan, respectively), and one experimental oat line "N979" (7.70% beta-glucan), were used to study the effect of beta-glucan levels in oat flours during simulated in vitro digestion and fermentation with human fecal flora obtained from different individuals. The oat flours were digested by using human digestion enzymes and fermented by batch fermentation under anaerobic conditions for 24 h. The fermentation progress was monitored by measuring pH, total gas, and short-chain fatty acid (SCFA) production. Significant effects of beta-glucan on the formation of gas and total SCFA were observed compared to the blank without substrate (P < 0.05); however, there were no differences in pH changes, total gas, and total SCFA production among oat lines (P > 0.05). Acetate, propionate, and butyrate were the main SCFA produced from digested oat flours during fermentation. More propionate and less acetate were produced from digested oat flours compared to lactulose. Different human fecal floras obtained from three healthy individuals had similar patterns in the change of pH and the production of gas during fermentation. Total SCFA after 24 h of fermentation were not different, but the formation rates of total SCFA differed between individuals. In vitro fermentation of digested oat flours with beta-glucan could provide favorable environmental conditions for the colon and these findings, thus, will help in developing oat-based food products with desirable health benefits.

Structure and function of starch and resistant starch from corn with different doses of mutant amylose-extender and floury-1 alleles.

Four corn types with different doses of mutant amylose-extender (ae) and floury-1 (fl1) alleles, in the endosperm, including no. 1, aeaeae; no. 2, fl1fl1fl1; no. 3, aeaefl1; and no. 4, fl1fl1ae, were developed for use in making Hispanic food products with high resistant starch (RS) content. The RS percentages in the native starch (NS) of 1-4 were 55.2, 1.1, 5.7, and 1.1%, respectively. All NS were evaluated for pasting properties with a rapid viscoanalyzer (RVA) and for thermal properties with a differential scanning calorimeter (DSC). NS 1 had a low peak viscosity (PV) caused by incomplete gelatinization, whereas NS 3 had the greatest PV and breakdown of all four starch types. On the DSC, NS 2 had the lowest onset temperature and greatest enthalpy. NS 1 and 3 had similar onset and peak temperatures, both higher than those of NS 2 and 4. The gel strength of NS heated with a RVA was evaluated by using a texture analyzer immediately after RVA heating (fresh, RVA-F) and after the gel had been stored at 4 degrees C for 10 days (retrograded, RVA-R). NS 1 gel was watery and had the lowest strength (30 g) among starch gel types. NS 3 gel, although exhibiting syneresis, had greater gel strength than NS 2 and 4. The structures of the NS, the RS isolated from the NS (RS-NS), the RS isolated from RVA-F (RS-RVA-F), and the RS isolated from RVA-R (RS-RVA-R) were evaluated by using size exclusion chromatography. NS 1 had a greater percentage of amylose (AM) (58.3%) than the other NS (20.4-26.8%). The RS from all NS types (RS-NS) had a lower percentage of amylopectin (AP) and a greater percentage of low molecular weight (MW) AM than was present in the original NS materials. The RS-RVA-R from all starches had no AP or high MW AM. The percentages of longer chain lengths (DP 35-60) of NS were greater in 1 and 3 than in 2 and 4, and the percentages of smaller chain lengths (DP 10-20) were greater in 2 and 4 than in 1 and 3. In general, NS 3 seemed to have inherited some pasting, thermal, and structural characteristics from both NS 1 and 2, but was distinctly different from 4.

Treatment of bruxism in individuals with developmental disabilities: a systematic review.

We reviewed studies involving the treatment of bruxism (i.e., teeth clenching or teeth grinding) in individuals with developmental disabilities. Systematic searches of electronic databases, journals, and reference lists identified 11 studies meeting the inclusion criteria. These studies were evaluated in terms of: (a) participants, (b) procedures used to assess bruxism, (c) intervention procedures, (d) results of the intervention, and (e) certainty of evidence. Across the 11 studies, intervention was provided to a total of 19 participants aged 4-43 years. Assessment procedures included dental screening under sedation and interviews with caregivers. Intervention approaches included prosthodontics, dental surgery, injection of botulinum toxin-a, behavior modification, music therapy, and contingent massage. Positive outcomes were reported in 82% of the reviewed studies. Overall, the evidence base is extremely limited and no definitive statements regarding treatment efficacy can be made. However, behavior modification and dental or medical treatment options (e.g., prosthodontics) seem to be promising treatment approaches. At present, a two-step assessment process, consisting of dental screening followed by behavioral assessment, can be recommended.

Treatment of elopement in individuals with developmental disabilities: a systematic review.

We reviewed studies involving the treatment of elopement in individuals with developmental disabilities. Systematic searches of three electronic databases, journals, and reference lists identified 10 studies meeting the inclusion criteria. These studies were evaluated in terms of: (a) participants, (b) procedures used to assess elopement, (c) intervention procedures, (d) results of the intervention, and (e) certainty of evidence. Across the 10 studies, intervention was provided to a total of 53 participants aged 3-47 years. Assessment procedures included anecdotal staff reports, participant interviews, direct observation, and modified analog functional analysis. Intervention approaches included differential reinforcement, extinction, functional communication training, response blocking, non-contingent reinforcement, shaping, and scheduled exercise. Positive outcomes were reported in 80% of the reviewed studies. The evidence base suggests that function-based assessment (e.g. functional analysis procedures) and function-based treatments (e.g. functional communication training) may be most effective in the treatment of elopement in this population. Directions for future research are offered.

Impact of dry solids and bile acid concentrations on bile acid binding capacity of extruded oat cereals.

Extruded breakfast cereals (EBC), processed from two oat lines, N979-5-2-4 (N979) and "Jim", with beta-glucan concentrations of 8.7 and 4.9%, respectively, were used to determine the impact of dry solids (DS) and bile acid (BA) concentrations on in vitro BA binding efficiency. A full fractional factorial design with levels for BA concentrations of 0.20, 0.47, 0.95, 2.37, and 4.73 micromol/g of total EBC slurry and for DS in the slurries of 0.8, 2, 3, and 4% (w/w) was selected. The absolute amount of BA bound (micromol) was measured for each trial in the experiment design. The percentage (%) of BA bound based on the total amount of BA added and BA bound per gram of DS of the EBC (micromol/g) were also presented and discussed. N979 in vitro digestion slurries had greater BA binding (micromol) than Jim slurries at different DS and BA concentrations, with greater differences at DS of 3% or above and at BA concentrations of 2.37 micromol/g or above. No difference in the absolute amount of BA bound (micromol) and percentage (%) BA bound occurred between the EBC slurries made from the two oat types at the lowest DS of 0.8% or the lowest BA concentration of 0.20 micromol/g. The efficiency of BA binding by beta-glucan in these two EBC became more distinguishable at 3% DS or above and BA concentrations of 2.37 micromol/g or above, indicating that these two conditions can be employed to measure BA capacities for similar foods. Also, the beta-glucan in the EBC produced from the N979 oat line was more soluble than that from the EBC produced from the Jim oat line. Thus, greater BA binding capacity may have been caused by both a greater amount of beta-glucan and a greater solubility of beta-glucan in N979 than in Jim EBC.

Digestion residues of typical and high-beta-glucan oat flours provide substrates for in vitro fermentation.

In vitro fermentabilities of the oat flour digestion residues (ODR) from two commercial oat lines with 4.7 and 5.3% beta-glucan and from two high-beta-glucan experimental lines with 7.6 and 8.1% beta-glucan were evaluated and compared with fermentations of lactulose, purified oat beta-glucan (POBG), and purified oat starch (POS). Substrates were fermented by using an in vitro batch fermentation system under anaerobic conditions for 24 h. The progress of the fermentation was studied by following the change in pH of the fermentation medium, production of short-chain fatty acids (SCFA) and gases, and consumption of carbohydrates. The substrate from the flour with the greatest amount of beta-glucan tended to have the greatest pH decline and the greatest total SCFA production. A significant correlation occurred between gas production and SCFA formation (R 2 = 0.89-0.99). Acetate was produced in the greatest amounts by all of the substrates except POBG, by which butyrate was produced in the greatest amount. More propionate and butyrate, but less acetate, were produced from high-beta-glucan ODR. With the given fermentation conditions, >80% of the total carbohydrate was depleted by the bacteria after 24 h. Glucose was the most rapidly consumed carbohydrate among other available monosaccharides in the fermentation medium. Overall, the high-beta-glucan experimental lines provided the best conditions for optimal in vitro gut fermentations.

A role for Akt in epidermal growth factor-stimulated cell cycle progression in cultured hepatocytes: generation of a hyperproliferative window after adenoviral expression of constitutively active Akt.

Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.