Protein Expression of PTTG1 as a Diagnostic Biomarker in Adrenocortical Carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) has a poor prognosis and there is an unmet clinical need for biomarkers to improve both diagnostic and prognostic assessment. Pituitary-tumor transforming gene (PTTG1) has been shown to modulate cancer invasiveness and response to therapy. The potential role of PTTG1 protein levels in ACC has not been previously addressed. We assessed whether increased nuclear protein expression of PTTG1 distinguished ACCs from adrenocortical adenomas (ACAs). METHODS: Patients with ACC or ACA were identified from prospective tissue banks at two independent institutions. Two tissue microarrays (TMAs) consisting of adrenal specimens from 131 patients were constructed and clinically annotated. Immunohistochemical analysis for PTTG1 and Ki-67 was performed on each TMA. RESULTS: TMA-1 (n = 80) contained 20 normal adrenals, 20 ACAs, and 40 ACCs, and the validation, TMA-2 (n = 51), consisted of 10 normal adrenals, 14 ACAs, and 27 ACCs. On TMA-1, nuclear staining of PTTG1 was detected in 12 (31%) ACC specimens, while all ACAs and normal adrenal glands were negative for PTTG1. On TMA-2, 20 (74%) of the ACC tumors demonstrated PTTG1 nuclear staining of PTTG1, and 13 (93%) ACA and 4 (44%) normal adrenal glands were negative for PTTG1. ACC tumors with increased PTTG1 protein staining had a significantly higher Ki-67 index (p < 0.001) than those with lower levels of PTTG1. CONCLUSIONS: Increased nuclear protein expression of PTTG1 was observed in malignant adrenal tumors. PTTG1 correlated with Ki-67 in two independent TMAs. PTTG1 is a promising biologic marker in the evaluation of adrenal tumors.

Clinical outcomes of cT1 micropapillary bladder cancer.

PURPOSE: While many urologists recommend radical cystectomy for micropapillary bladder cancer invading the lamina propria (cT1), contradictory small reports exist on the efficacy of conservative management with intravesical bacillus Calmette-Guérin for this disease. We report our updated experience in what to our knowledge is the largest series of patients with cT1 micropapillary bladder cancer. MATERIALS AND METHODS: An institutional review board approved review of our cancer database identified 283 patients with micropapillary bladder cancer, including 72 staged with cT1N0M0 disease at diagnosis and initiation of therapy. Survival analysis was performed using the Kaplan-Meier estimator and compared using the log rank test. RESULTS: In this cohort of 72 patients 40 received primary intravesical bacillus Calmette-Guérin and 26 underwent up-front radical cystectomy. Of patients who received bacillus Calmette-Guérin 75%, 45% and 35% experienced disease recurrence, progression and lymph node metastasis, respectively. Patients treated with up-front cystectomy had improved survival compared to patients treated with primary bacillus Calmette-Guérin (5-year disease specific survival 100% vs 60% p = 0.006) and patients who underwent delayed cystectomy after recurrence (5-year disease specific survival 62%, p = 0.015). Prognosis was especially poor in patients who waited for progression before undergoing radical cystectomy with an estimated 5-year disease specific survival of only 24% and a median survival of 35 months. In patients treated with up-front cystectomy pathological up-staging was found in 27%, including 20% with lymph node metastasis. CONCLUSIONS: While certain patients with T1 micropapillary bladder cancer may respond to intravesical bacillus Calmette-Guérin, survival is improved in those who undergo early radical cystectomy. Further molecular studies are needed to identify subsets of patients in whom the bladder can be safely spared.

Detection of mitotic figures and G2+ tumor nuclei with histone markers correlates with worse overall survival in patients with Merkel cell carcinoma.

BACKGROUND: High mitotic figure count (MFC) correlates with low survival rate in Merkel cell carcinoma (MCC). However, the prognostic impact of histone biomarkers as surrogates of MFC in MCC is unknown. We evaluated the prognostic significance of the immunodetection of mitotic figures and of G2+ tumor nuclei with histone-associated mitotic markers H3K79me3T80ph (H3KT) and phosphohistone H3 (PHH3) in MCC. METHODS: Immunohistochemical analyses of H3KT and PHH3 and proliferative marker Ki-67 were performed in a series of 21 cases of MCC. The significance of the pathologic data and immunoreactivity with these markers was evaluated with Pearson correlation and paired Student t-test. Univariate Cox proportional hazards regression models were performed to assess the relationships between these markers and survival. RESULTS: H3KT detected a higher number of mitotic figure (p<0.0001) and G2+ tumor nuclei (p<0.0052) than did PHH3. Furthermore, the MFC combined with G2+ tumor nuclei detected with H3KT compared to PHH3 and manual MFC was a significant predictor of impaired survival in patients with MCC (p=0.035; HR=1.0172), corresponding to a 1.72% increased risk of death for each unit increase in H3KT. CONCLUSIONS: Biomarker analysis of proliferative rates with histone markers may have relevance in stratifying risk in patients with MCC.

Single-Cell and Spatial Analysis of Emergent Organoid Platforms.

Organoids have emerged as a promising advancement of the two-dimensional (2D) culture systems to improve studies in organogenesis, drug discovery, precision medicine, and regenerative medicine applications. Organoids can self-organize as three-dimensional (3D) tissues derived from stem cells and patient tissues to resemble organs. This chapter presents growth strategies, molecular screening methods, and emerging issues of the organoid platforms. Single-cell and spatial analysis resolve organoid heterogeneity to obtain information about the structural and molecular cellular states. Culture media diversity and varying lab-to-lab practices have resulted in organoid-to-organoid variability in morphology and cell compositions. An essential resource is an organoid atlas that can catalog protocols and standardize data analysis for different organoid types. Molecular profiling of individual cells in organoids and data organization of the organoid landscape will impact biomedical applications from basic science to translational use.

Modeling digestion, absorption, and ketogenesis after administration of tricaprilin formulations to humans.

At present, tricaprilin is used as a ketogenic source for the management of mild to moderate Alzheimer's disease. After administration of the medium-chain triglyceride, tricaprilin is hydrolyzed to octanoic acid and further metabolized to ketones, acting as an alternative energy substrate for the brain. In this investigation, we developed a physiologically-based biopharmaceutics model simulating in vivo processes following the peroral administration of tricaprilin. The model includes multiple data sources to establish a partially verified framework for the simulation of plasma profiles. The input parameters were identified based on existing literature data and in vitro digestion studies. Model validation was conducted using the data from a phase I clinical trial. A partial parameter sensitivity analysis elucidated various influences on the plasma ketone levels that are mainly responsible for the therapeutic effects of tricaprilin. Based on our findings, we concluded that dispersibility and lipolysis of tricaprilin together with the gastric emptying patterns are limiting ketogenesis, while other steps such as the conversion of octanoic acid to ketone bodies play a minor role only.

Imaging high jitter, very fast phenomena: A remedy for shutter lag.

Dielectric breakdown is an example of a natural phenomenon that occurs on very short time scales, making it incredibly difficult to capture optical images of the process. Event initiation jitter is one of the primary challenges, as even a microsecond of jitter time can cause the imaging attempt to fail. Initial attempts to capture images of dielectric breakdown using a gigahertz frame rate camera and an exploding bridge wire initiation were stymied by high initiation jitter. Subsequently, a novel optical delay line apparatus was developed in order to effectively circumvent the jitter and reliably image dielectric breakdown. The design and performance of the optical delay line apparatus are presented. The optical delay line increased the image capture success rate from 25% to 94% while also permitting enhanced temporal resolution and has application in imaging other high-jitter, extremely fast phenomena.

Pharmacogenetic analysis of the effects of polymorphisms in APOE, IDE and IL1B on a ketone body based therapeutic on cognition in mild to moderate Alzheimer's disease; a randomized, double-blind, placebo-controlled study.

BACKGROUND: To examine the effect of genetic variation in APOE, IDE and IL1B on the response to induced ketosis in the Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) in subjects with mild to moderate Alzheimer's disease (AD). METHODS: Genotype effects on ADAS-Cog scores from a randomized, double-blind, placebo-controlled study in mild to moderate AD were examined by an overall two way analysis of variance. In addition, interactions with the carriage status of the epsilon 4 allele of the APOE gene (APOE4) were examined. RESULTS: Significant differences in response to induced ketosis were found among non-carriers of putative gain-of-function polymorphisms in rs1143627 and rs16944 in the IL1B gene and among variants of the polymorphism rs2251101 in the IDE gene. Significant differences were found among non-carriers of the APOE4 gene, with notable improvement among the E3/E3 genotype group. CONCLUSIONS: Variants in APOE, IL1B and IDE may influence the cognitive response to induced ketosis in patients with mild to moderate AD. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov, registry number NCT00142805.

A Placebo-Controlled, Parallel-Group, Randomized Clinical Trial of AC-1204 in Mild-to-Moderate Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by amyloid-ß plaques, neurofibrillary tangles, and regional cerebral glucose hypometabolism. Providing an alternative metabolic substrate, such as ketone bodies, may be a viable therapeutic option. OBJECTIVE: The objective was to determine the efficacy and safety of the AC-1204 formulation of caprylic triglyceride administered daily for 26 weeks in APOE4 non-carrier participants with mild-to-moderate AD. METHODS: In a double-blind, placebo-controlled, randomized study (AC-12-010, NOURISH AD, NCT01741194), 413 patients with mild-to-moderate probable AD were stratified by APOE genotype and randomized (1 : 1) to receive either placebo or AC-1204 for 26 weeks. The primary outcome was the change from baseline to week 26 on the 11-item Alzheimer's Disease Assessment Scale - Cognitive subscale (ADAS-Cog11) among APOE4 non-carriers. The key secondary outcome was the change from baseline to week 26 in the Alzheimer's Disease Cooperative Study - Clinician's Global Impression of Change scale. RESULTS: Administration of AC-1204 was safe and well-tolerated. Mean changes from baseline in the primary outcome at 26 weeks in ADAS-Cog11 for placebo (n = 138) was 0.0 and for AC-1204 (n = 137) was 0.6 (LS differences of mean - 0.761, p = 0.2458) and secondary outcome measures failed to detect any drug effects. CONCLUSION: The AC-1204 formulation of caprylic triglyceride failed to improve cognition or functional ability in subjects with mild-to-moderate AD. The lack of efficacy observed in this study may have several contributing factors including a lower ketone body formation from AC-1204 than expected and a lack of decline in the patients receiving placebo.

Cytogenetic and molecular characterization of a partial trisomy 2p arising from inverted duplication of 2p with terminal deletion of 2pter.

Partial trisomy 2p is typically associated with partial monosomy of another chromosomal segment and results from a balanced translocation in one of the parents. Inverted duplications with terminal deletions have been reported in an increasing number of chromosomes. Several cases initially interpreted as terminal duplications have instead been documented to represent inverted duplications with terminal deletions. Inv dup del(2p) has been reported in patients who manifest the clinical findings of trisomy 2p syndrome. Here we report on a 2-month-old girl with inv dup del(2p) and clinical manifestations that overlap those found commonly in partial 2p trisomy, as previously reported in the literature. Her clinical picture helps delineate the phenotype of 2p duplication disorders.

Dietary enrichment with medium chain triglycerides (AC-1203) elevates polyunsaturated fatty acids in the parietal cortex of aged dogs: implications for treating age-related cognitive decline.

Dogs demonstrate an age-related cognitive decline, which may be related to a decrease in the concentration of omega-3 polyunsaturated fatty acids (n-3 PUFA) in the brain. Medium chain triglycerides (MCT) increase fatty acid oxidation, and it has been suggested that this may raise brain n-3 PUFA levels by increasing mobilization of n-3 PUFA from adipose tissue to the brain. The goal of the present study was to determine whether dietary MCT would raise n-3 PUFA concentrations in the brains of aged dogs. Eight Beagle dogs were randomized to a control diet (n = 4) or an MCT (AC-1203) enriched diet (n = 4) for 2 months. The animals were then euthanized and the parietal cortex was removed for phospholipid, cholesterol and fatty acid determinations by gas-chromatography. Dietary enrichment with MCT (AC-1203) resulted in a significant increase in brain phospholipid and total lipid concentrations (P < 0.05). In particular, n-3 PUFA within the phospholipid, unesterified fatty acid, and total lipid fractions were elevated in AC-1203 treated subjects as compared to controls (P < 0.05). Brain cholesterol concentrations did not differ significantly between the groups (P > 0.05). These results indicate that dietary enrichment with MCT, raises n-3 PUFA concentrations in the parietal cortex of aged dogs.

Hypometabolism as a therapeutic target in Alzheimer's disease.

The pathology of Alzheimer's disease (AD) is characterized by cerebral atrophy in frontal, temporal, and parietal regions, with senile plaques, dystrophic neurites, and neurofibrillar tangles within defined areas of the brain. Another characteristic of AD is regional hypometabolism in the brain. This decline in cerebral glucose metabolism occurs before pathology and symptoms manifest, continues as symptoms progress, and is more severe than that of normal aging. Ketone bodies are an efficient alternative fuel for cells that are unable to metabolize glucose or are 'starved' of glucose. AC-1202 is designed to elevate serum ketone levels safely. We previously showed that treatment with AC-1202 in patients with mild-to-moderate AD improves memory and cognition. Treatment outcomes were influenced by apolipoprotein E genotype status. These data suggest that AC-1202 may be an effective treatment for cognitive dysfunction by providing an alternative substrate for use by glucose-compromised neurons.

Induction of ketosis may improve mitochondrial function and decrease steady-state amyloid-beta precursor protein (APP) levels in the aged dog.

Region specific declines in the cerebral glucose metabolism are an early and progressive feature of Alzheimer's disease (AD). Such declines occur pre-symptomatically and offer a potential point of intervention in developing AD therapeutics. Medium chain triglycerides (MCTs), which are rapidly converted to ketone bodies, were tested for their ability to provide an alternate energy source to neurons suffering from compromised glucose metabolism. The present study determined the short-term effects of ketosis in aged dogs, a natural model of amyloidosis. The animals were administered a 2 g/kg/day dose of MCTs for 2 months. Mitochondrial function and oxidative damage assays were then conducted on the frontal and parietal lobes. Amyloid-beta (Abeta), amyloid precursor protein (APP) processing and beta-site APP cleaving enzyme (BACE1) assays were conducted on the frontal, parietal and occipital lobes. Aged dogs receiving MCTs, as compared to age-matched controls, showed dramatically improved mitochondrial function, as evidenced by increased active respiration rates. This effect was most prominent in the parietal lobe. The improved mitochondrial function may have been due to a decrease in oxidative damage, which was limited to the mitochondrial fraction. Steady-state APP levels were also decreased in the parietal lobe after short-term MCT administration. Finally, there was a trend towards a decrease in total Abeta levels in the parietal lobe. BACE1 levels remained unchanged. Combined, these findings suggest that short-term MCT administration improves energy metabolism and decreases APP levels in the aged dog brain.

Bioaccessibility of U, Th and Pb in solid wastes and soils from an abandoned uranium mine.

Bioaccessible U, Th, Pb and the 238U decay products 214Pb and 210Pb have been determined, using a modified Unified BARGE Method (UBM), in waste solids and soils from an abandoned uranium mine in South West England, UK. Maximum aqua regia extractable concentrations for U, Th and Pb were 16,200, 3.8 and 4750 µg g-1, respectively. 238U had highest activity concentrations near the mine shaft, where the decay products214Pb and 210Pb had values of 235 and 180 Bq g-1, respectively. UBM extractions gave mean gastro-intestinal bioaccessibility factors (BAFs) for U and Pb in the waste solids of 0.05 and 0.03, respectively, whereas those for the soils were significantly higher at 0.24 and 0.17. The mean BAFs for the transient radionuclides, 214Pb and 210Pb, were similar to those for stable Pb implying that the stable and radioactive Pb isotopes were attached to similar sites on the particles. The doses arising from the ingestion of particulate 210Pb due to soil pica behaviour were in the range 0.2-65 and < 0.1-6.2 µSv day-1 for a 1-year old child or an adult (>17 years), respectively. The results suggest that the health risk posed by abandoned uranium mines, with waste rock and tailings, throughout the world should take account of the dose due to both bioaccessible radionuclides, as well as their stable counterparts.

Reduced expression of frataxin extends the lifespan of Caenorhabditis elegans.

Defects in the expression of the mitochondrial protein frataxin cause Friedreich's ataxia, an hereditary neurodegenerative syndrome characterized by progressive ataxia and associated with reduced life expectancy in humans. Homozygous inactivation of the frataxin gene results in embryonic lethality in mice, suggesting that frataxin is required for organismic survival. Intriguingly, the inactivation of many mitochondrial genes in the nematode Caenorhabditis elegans by RNAi extends lifespan. We therefore investigated whether inactivation of frataxin by RNAi-mediated suppression of the frataxin homolog gene (frh-1) would also prolong lifespan in the nematode. Frataxin-deficient animals have a small body size, reduced fertility and altered responses to oxidative stress. Importantly, frataxin suppression by RNAi significantly extends lifespan in C. elegans.

daf-16 protects the nematode Caenorhabditis elegans during food deprivation.

Inhibition of either the insulin-like or target of rapamycin (TOR) pathways in the nematode Caenorhabditis elegans extends life span. Here, we demonstrate that starvation and inhibition of the C. elegans insulin receptor homolog (daf-2) elicits a daf-16-dependent up-regulation of a mitochondrial superoxide dismutase (sod-3). We also find that although heat and oxidative stress result in nuclear localization of the DAF-16 protein, these stressors do not activate a SOD-3 reporter, suggesting that nuclear localization alone may not be sufficient for transcriptional activation of DAF-16. We show that inhibition of either TOR activity or key components of the cognate translational machinery (eIF-4G and EIF-2B homologs) increases life span by both daf-16-dependent and -independent mechanisms. Finally, we demonstrate that at least one nematode hexokinase is localized to the mitochondria. We propose that the increased life spans conferred by alterations in both the TOR and insulin-like pathways function by inappropriately activating food-deprivation pathways.

Effects of beta-hydroxybutyrate on cognition in memory-impaired adults.

Glucose is the brain's principal energy substrate. In Alzheimer's disease (AD), there appears to be a pathological decrease in the brain's ability to use glucose. Neurobiological evidence suggests that ketone bodies are an effective alternative energy substrate for the brain. Elevation of plasma ketone body levels through an oral dose of medium chain triglycerides (MCTs) may improve cognitive functioning in older adults with memory disorders. On separate days, 20 subjects with AD or mild cognitive impairment consumed a drink containing emulsified MCTs or placebo. Significant increases in levels of the ketone body beta-hydroxybutyrate (beta-OHB) were observed 90 min after treatment (P=0.007) when cognitive tests were administered. beta-OHB elevations were moderated by apolipoprotein E (APOE) genotype (P=0.036). For 4+ subjects, beta-OHB levels continued to rise between the 90 and 120 min blood draws in the treatment condition, while the beta-OHB levels of 4- subjects held constant (P<0.009). On cognitive testing, MCT treatment facilitated performance on the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog) for 4- subjects, but not for 4+ subjects (P=0.04). Higher ketone values were associated with greater improvement in paragraph recall with MCT treatment relative to placebo across all subjects (P=0.02). Additional research is warranted to determine the therapeutic benefits of MCTs for patients with AD and how APOE-4 status may mediate beta-OHB efficacy.

High carbohydrate diets and Alzheimer's disease.

Alzheimer's disease (AD) is a common, progressive, neurodegenerative disease that primarily afflicts the elderly. A well-defined risk factor for late onset AD is possession of one or more alleles of the epsilon-4 variant (E4) of the apolipoprotein E gene. Meta-analysis of allele frequencies has found that E4 is rare in populations with long historical exposure to agriculture, suggesting that consumption of a high carbohydrate (HC) diet may have selected against E4 carriers. The apoE4 protein alters lipid metabolism in a manner similar to a HC diet, suggesting a common mechanism for the etiology of AD. Evolutionarily discordant HC diets are proposed to be the primary cause of AD by two general mechanisms. (1) Disturbances in lipid metabolism within the central nervous system inhibits the function of membrane proteins such as glucose transporters and the amyloid precursor protein. (2) Prolonged excessive insulin/IGF signaling accelerates cellular damage in cerebral neurons. These two factors ultimately lead to the clinical and pathological course of AD. This hypothesis also suggests several preventative and treatment strategies. A change in diet emphasizing decreasing dietary carbohydrates and increasing essential fatty acids (EFA) may effectively prevent AD. Interventions that restore lipid homeostasis may treat the disease, including drugs that increase fatty acid metabolism, EFA repletion therapy, and ketone body treatment.