RNA-Seq Analysis of Differentiated Keratinocytes Reveals a Massive Response to Late Events during Human Papillomavirus 16 Infection, Including Loss of Epithelial Barrier Function.

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalized keratinocytes (NIKS) and NIKS stably transfected with HPV16 episomal genomes (NIKS16) were compared using next-generation sequencing (RNA-Seq). HPV16 infection altered expression of 2,862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNA-Seq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change), 670 genes were downregulated and 296 genes were upregulated. HPV downregulated many genes involved in epithelial barrier function, which involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant CCL20, and proinflammatory cytokines interleukin 1α (IL-1α) and IL-1ß. However, the type I interferon regulator IRF1, kappa interferon (IFN-κ), and viral restriction factors (IFIT1, -2, -3, and -5, OASL, CD74, and RTP4) were upregulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions, and cell adhesion. Quantitative PCR (qRT-PCR) and Western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE HPV genome amplification and capsid formation take place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNA-Seq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3,000 genes were differentially expressed in keratinocytes due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into the virus-host interaction that is crucial for the production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.

Preliminary Investigation of Cerebral Blood Flow and Amyloid Burden in Veterans With and Without Combat-Related Traumatic Brain Injury.

This study aimed to examine global and regional cerebral blood flow and amyloid burden in combat veterans with and without traumatic brain injury (TBI). Cerebral blood flow (in milliliters per minute per 100 mL) was measured by quantitative [(15)O]water, and amyloid burden was measured by [(11)C]PIB imaging. Mean global cerebral blood flow was significantly lower in veterans with TBI compared with non-TBI veterans. There were essentially no differences between groups for globally normalized regional cerebral blood flow. Amyloid burden did not differ between TBI and non-TBI veterans. Veterans who have suffered a TBI have significantly lower cerebral blood flow than non-TBI controls but did not manifest increased levels of amyloid, globally or regionally.

30 years of [11C]methyl triflate.

Some scientific discoveries are well known only to a core group of researchers working on technical subjects. Nevertheless, they open new research directions, allow existing knowledge to be viewed in entirely new and useful ways, or provide a way to make something that was hard or impossible to make before. Carbon-11 methyl triflate ([11C]MeOTf) is one such advance, facilitating the synthesis of many carbon-11 radio tracers and broadening the range of applications of carbon-11 radiochemistry. The year 2022 marked the 30th anniversary of the original paper in Applied Radiation and Isotopes introducing a simple synthesis of [11C]MeOTf from carbon-11 methyl iodide ([11C]MeI) and it also marked the end of the fruitful career and life of the researcher who developed it, Douglas Jewett. It seems fitting to say a few words on how it came to be and how it has helped advance carbon-11 radiochemistry.

Acute ill-health in sheep farmers following use of pesticides.

BACKGROUND: Sheep farmers often complain of acute ill-health, known colloquially as 'dipper's flu', immediately after treating sheep with pesticides. There have been few prospective epidemiological studies to determine it's nature and incidence. Aims To determine the nature and frequency of symptoms occurring in farmers treating sheep for ectoparasites. METHODS: In a longitudinal study, farmers who planned to treat their sheep for ectoparasites were recruited. Farmers kept a symptom diary for 7 days after starting pesticide treatment. Symptoms reported on days 1-6 were compared to those reported on day 7 via the McNemar's test and with previously published literature definitions of dipper's flu. A principal component analysis (PCA) was carried out on new symptoms occurring on days 1 and 2. RESULTS: Of 781 farmers recruited, 352 farmers (45%) completed the symptom diary. In the 7 days after starting pesticide treatment, symptom complex reporting typically peaked on day 2, but few farmers (7 or less; <2%) were identified as having dipper's flu using literature definitions. However, PCA identified two new patterns of symptom complexes that accounted for 35% of the variance. A pyrexial factor consisted of four symptom complexes (feeling generally ill; feeling sweaty, shivery, feverish, hot or cold; feeling unusually tired; and having a headache) and a respiratory factor consisted of three symptom complexes (runny, stuffy, blocked or irritated nose; cough, shortness of breath or wheeze; and eye irritation). CONCLUSIONS: Existing definitions of dipper's flu do not adequately describe symptoms that occur following the treatment of sheep for ectoparasites.

The application of membrane bioreactors as decentralised systems for removal of endocrine disrupting chemicals and pharmaceuticals.

The concentrations of some important endocrine disrupting chemicals and pharmaceuticals after various stages of wastewater treatment were investigated. The endocrine disrupting chemicals included natural and synthetic estrogenic and androgenic steroids. The pharmaceuticals included a series of sulfonamide antibiotics and trimethoprim. The removal efficiency of a membrane bioreactor (MBR) was investigated and compared with a conventional activated sludge (CAS) system. Samples were analysed by liquid chromatography tandem mass spectrometry. Results showed that the MBR and CAS systems effectively removed steroidal estrogens and androgens, but only partially eliminated the target antibiotics from wastewater. The MBR was shown to be more effective than the CAS system which was possibly attributed to the high solid retention time and concentration of biosolids in the MBR. The results highlight the potential wider application of MBRs for the removal of trace chemical contaminants in wastewater and their potential for use as decentralised wastewater treatment systems.

Kinetic analysis of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) in head and neck cancer patients before and early after initiation of chemoradiation therapy.

UNLABELLED: The purpose of this study was to investigate the kinetic behavior of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) before and early after initiation of chemoradiation therapy in patients with squamous cell head and neck cancer. METHODS: A total of 8 patients with head and neck cancer underwent (18)F-FLT PET scans (7 patients at baseline and after 5 d [10 Gy] of radiation therapy given with concomitant chemotherapy and 1 patient only at baseline). Dynamic PET images were obtained with concurrent arterial or venous blood sampling. Kinetic parameters including the flux constant of (18)F-FLT based on compartmental analysis (K-FLT), the Patlak influx constant (K-Patlak), and standardized uptake value (SUV) were calculated for the primary tumor and (18)F-FLT-avid cervical lymph nodes for all scans. RESULTS: Mean pretreatment values of uptake for the primary tumor and cervical nodes were 0.075 +/- 0.006 min(-1), 0.042 +/- 0.004 min(-1), and 3.4 +/- 0.5 (mean +/- SD) for K-FLT, K-Patlak, and SUV, respectively. After 10 Gy of radiation therapy, these values were 0.040 +/- 0.01 min(-1), 0.018 +/- 0.016 min(-1), and 1.8 +/- 1.1 for K-FLT, K-Patlak, and SUV, respectively. For all lesions seen on pretherapy and midtherapy scans, the correlation was 0.90 between K-FLT and K-Patlak, 0.91 between K-FLT and SUV, and 0.99 between K-Patlak and SUV. CONCLUSION: The initial (18)F-FLT uptake and change early after treatment in squamous head and neck tumors can be adequately characterized with SUV obtained at 45-60 min, which demonstrates excellent correlation with influx parameters obtained from compartmental and Patlak analyses.

The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.

OBJECTIVE: To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function. METHODS: Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion. CONCLUSIONS: We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

Fate and analysis of endocrine disrupting chemicals in some sewage treatment plants in Australia.

There are limited studies on the fate and levels of endocrine disrupting chemicals in sewage treatment plants in Australia. Research undertaken in Europe and North America has shown biologically significant levels of both oestrogenic and androgenic chemicals in sewage effluent. The aim of this work was to determine the oestrogenic and androgenic activities of raw and treated sewage from sewage treatment plants run by MidCoast Water, New South Wales, Australia. Oestrogenic and androgenic activities were measured using a yeast screen bioassay. Results showed that the raw effluent contained biologically significant levels of both oestrogenic (0.58-2.91 ng/l) and androgenic (216-480 ng/l) activities. Androgenic activity was significantly higher than oestrogenic activity, which was consistent with other Australian studies and was attributed to the higher levels of androgens in domestic waste from human excretion compared to oestrogens. Secondary treatment (using activated sludge) removed the majority of the oestrogenic and androgenic activity (up to 99%). Tertiary treatment by UV removed varying levels of oestrogenic (19-69%) and androgenic (5-55%) activities. A Membrane Bioreactor (MBR) at one of the STPs, which consists of an MBR followed by electrochlorination removed over 87% of the oestrogenic activity and over 98% of androgenic activity from raw sewage samples. However, levels which could be biologically significant still remained after secondary and tertiary treatment (>0.1 ng/l oestrogenic activity and >1 ng/l androgenic activity).

Optimizing the radiosynthesis of [68Ga]DOTA-MLN6907 peptide containing three disulfide cyclization bonds - a GCC specific chelate for clinical radiopharmaceuticals.

In the present study, the effect of radiolabeling conditions on radiolabeling efficiency and achievable specific activity of a DOTA-conjugated highly-lipophilic peptide containing three disulfide cyclization bonds was examined. The peptide is designed to bind specifically (with high affinity) to cell-surface receptor guanylyl cyclase C (GCC), which is universally expressed by colorectal cancer cells. The effect of systematic variation of chemical parameters pH, mass of peptide, acetate buffer concentration (ionic strength), and inclusion of ethanol in the radiolabeling reaction vessel on achievable specific activity and labeling efficiency was examined. In addition, a unique approach to acetone-based elution of 68Ga from an initial cation-exchange pre-concentration column is introduced, which improved radiochemical yield and radiochemical purity. For the evaluation of the acetone-based method, two different post-radiolabeling reverse-phase (C18) approaches to purify the final radiolabeled peptide were tested. These results revealed the potential for peptide degradation via the cleavage of disulfide cyclization bonds to form free thiols when using one of these C18 cartridges. The final optimized procedure enabled radiolabeling efficiency of greater than 99% and specific activity greater than 35 MBq/nmole in less than 30 min. The optimized parameters were amenable to the use of an automated 68Ge/68Ga generator and fluid-handling system for clinical production of the GCC receptor-specific [68Ga]DOTA-MLN6907 peptide. The chemical characteristics of individual peptides govern the most appropriate radiolabeling conditions for the preparation of radiopharmaceuticals.

90Y-DOTATOC Dosimetry-Based Personalized Peptide Receptor Radionuclide Therapy.

Pretherapy PET with 86Y-DOTATOC is considered the ideal dosimetry protocol for 90Y-DOTATOC therapy; however, its cost, limited availability, and need for infusion of amino acids to mimic the therapy administration limit its use in the clinical setting. The goal of this study was to develop a dosimetric method for 90Y-DOTATOC using 90Y-DOTATOC PET/CT and bremsstrahlung SPECT/CT and to determine whether dosimetry-based administered activities differ significantly from standard administered activities. Methods: This was a prospective phase 2 trial of 90Y-DOTATOC therapy in patients with somatostatin receptor-positive tumors. 90Y-DOTATOC was given in 3 cycles 6-8 wk apart. In the first cycle of therapy, adults received 4.4 GBq and children received 1.85 GBq/m2; the subsequent administered activities were adjusted according to the dosimetry of the preceding cycle so as not to exceed a total kidney dose of 23 Gy and bone marrow dose of 2 Gy. The radiation dose to the kidneys was determined from serial imaging sessions consisting of time-of-flight 90Y-DOTATOC PET/CT at 5 h after therapy and 90Y-DOTATOC bremsstrahlung SPECT/CT at 6, 24, 48, and 72 h. The PET/CT data were used to measure the absolute concentration of 90Y-DOTATOC and to calibrate the bremsstrahlung SPECT kidney clearance data. The radiation dose to the kidneys was determined by multiplying the time-integrated activity (from the fitted biexponential curve of renal clearance of 90Y-DOTATOC) with the energy emitted per decay, divided by the mass of the kidneys. Results: The radiation dose to the kidneys per cycle of 90Y-DOTATOC therapy was highly variable among patients, ranging from 0.32 to 3.0 mGy/MBq. In 17 (85%) of the 20 adult patients who received the second and the third treatment cycles of 90Y-DOTATOC, the administered activity was modified by at least 20% from the starting administered activity. Conclusion: Renal dosimetry of 90Y-DOTATOC is feasible using 90Y-DOTATOC time-of-flight PET/CT and bremsstrahlung SPECT/CT and has a significant impact on the administered activity in treatment cycles.

Expression of the WASP verprolin-homologues (WAVE members) in human breast cancer.

BACKGROUND: The WASP family proteins have been indicated to play a vital role in the formation of membrane protrusions required for cell locomotion. WAVE proteins are an important subfamily that also plays a crucial role in actin polymerisation, which is vital to cell migration. However, not much is known about the clinical significance of this subfamily in cancers. We report, for the first time, the expression of the WAVE molecules, at protein and mRNA levels, in human breast cancer. MATERIALS AND METHODS: The expression of the 3 WAVE molecules at the mRNA and protein levels in a cohort of 122 human breast cancers and 32 normal breast tissues were analysed and correlated with the patients' pathological and clinical information as well as outcome (120 months follow-up). RESULTS: All 3 WAVE molecules were detected in mammary tissues. WAVE2 transcripts were expressed in high levels in all breast tumours. Over-expression of WAVE2 was seen in node-positive cases as well as in moderately and poorly differentiated tumours. Also, high levels of WAVE2 expression were associated with death due to disease (p = 0.02) at follow-up. No distinct associations were found between the WAVE1 and WAVE3 transcripts and the breast cancer cells.

Pharmacoimaging of Blood-Brain Barrier Permeable (FDG) and Impermeable (FLT) Substrates After Intranasal (IN) Administration.

To illustrate the use of imaging to quantify the transfer of materials from the nasal cavity to other anatomical compartments, specifically, transfer to the brain using the thymidine analogue, [18F]fluorothymidine (FLT), and the glucose analogue, [18F]fluorodeoxyglucose (FDG). Anesthetized rats were administered FLT or FDG by intranasal instillation (IN) or tail-vein injection (IV). PET/CT imaging was performed for up to 60 min. Volumes-of-interest (VOIs) for the olfactory bulb (OB) and the remaining brain were created on the CT and transferred to the co-registered dynamic PET. Time-activity curves (TACs) were generated and compared. The disposition patterns were successfully visualized and quantified and differences in brain distribution patterns were observed. For FDG, the concentration was substantially higher in the OB than the brain only after IN administration. For FLT, the concentration was higher in the OB than the brain after both IN and IV and higher after IN than after IV administration at all times, whereas the concentration in the brain was higher after IN than after IV administration at early times only. Approximately 50 and 9% of the IN FDG and FLT doses, respectively, remained in the nasal cavity at 20 min post-administration. The initial phase of clearance was similar for both agents (t1/2 = 2.53 and 3.36 min) but the slow clearance phase was more rapid for FLT than FDG (t1/2 = 32.1 and 85.2 min, respectively). Pharmacoimaging techniques employing PET/CT can be successfully implemented to quantitatively investigate and compare the disposition of radiolabeled agents administered by a variety of routes.

Localization of Unknown Primary Site with 68Ga-DOTATOC PET/CT in Patients with Metastatic Neuroendocrine Tumor.

Localization of the site of the unknown primary tumor is critical for surgical treatment of patients presenting with neuroendocrine tumor (NET) with metastases. Methods: Forty patients with metastatic NET and unknown primary site underwent 68Ga-DOTATOC PET/CT in a single-site prospective study. The 68Ga-DOTATOC PET/CT was considered true-positive if the positive primary site was confirmed by histology or follow-up imaging. The scan was considered false-positive if no primary lesion was found corresponding to the 68Ga-DOTATOC-positive site. All negative scans for primary tumor were considered false-negative. A scan was classified unconfirmed if 68Ga-DOTATOC PET/CT suggested a primary, however, no histology was obtained and imaging follow-up was not confirmatory. Results: The true-positive, false-positive, false-negative, and unconfirmed rates for unknown primary tumor were 38%, 7%, 50%, and 5%, respectively. Conclusion:68Ga-DOTATOC PET/CT is an effective modality in the localization of unknown primary in patients with metastatic NET.

Di-methyl-ammonium 2,4,5-tri-carb-oxy-benzoate: an example of the deca-rbonylation of N,N-di-methyl-formamide in the presence of a metal and a benzene-polycarb-oxy-lic acid. Is zirconium(IV) the Tsotsi?

The title salt, C2H8N+·C10H5O8-, was the unexpected product of an attempt to prepare a ZrIV metal-organic framework with benzene-1,2,4,5-tetra-carb-oxy-lic acid (1,2,4,5-H3B4C). In the reaction, the DMF solvent has been decarb-on-yl-ated, forming the di-methyl-ammonium cation, with one proton lost from the tetra-carb-oxy-lic acid. It is proposed that the ZrIV salt acts as a Tsotsi or robber, plundering CO from the DMF mol-ecule. The resulting salt crystallizes with two cations and two anions in the asymmetric unit. An intra-molecular hydrogen bond forms between a carb-oxy-lic acid substituent and the carboxyl-ate group of each of the monodeprotonated (1,2,4,5-H3B4C-) anions. In the crystal, an extensive array of O-H⋯O, N-H⋯O and C-H⋯O hydrogen bonds generates a three-dimensional network, with columns of cations and anions forming along the b axis.

Auditory attentional deficits in patients with schizophrenia. A positron emission tomography study.

BACKGROUND: Patients with schizophrenia have frequently been found to perform poorly on tasks requiring selective attention, defined as the ability to focus attention on relevant information while simultaneously ignoring irrelevant stimuli. This study explores the brain mechanisms mediating attentional processing in patients with schizophrenia by measuring their regional cerebral blood flow (rCBF) with positron emission tomography using [15O] water as they performed tasks that differed systematically in attentional demand. METHODS: Ten schizophrenic patients (either neurolepticnaive or withdrawn from medication) (patient group) and 10 normal volunteers (control group) performed auditory target detection tasks. Different types of auditory stimuli (environmental sounds, meaningless speech sounds, and words) were presented either binaurally (ie, same sounds in both ears) or dichotically (simultaneous and different sounds in the 2 ears). In dichotic conditions, subjects were instructed to focus on either their left or right ear. RESULTS: Initial subtraction-based image analyses sought significant rCBF changes anywhere in the brain. Patients consistently had less significant activation than controls in right superotemporal gyrus (STG). Follow-up analyses used regions of interest traced on individual magnetic resonance images to precisely measure rCBF in STG. Unlike controls, patients had higher rCBF in the left STG during all activation conditions. CONCLUSIONS: The abnormal task-related rCBF asymmetry in STG of schizophrenic patients may indicate an isolated temporal lobe deficit, but it may also indicate abnormality in the thalamocortical circuitry mediating selective attention and/or in the brain systems that integrate auditory processing in the 2 hemispheres.

The Coxsackie-adenovirus receptor has elevated expression in human breast cancer.

The Coxsackie-adenovirus receptor (CAR) is the primary site for adenovirus attachment during infection and has been used as a delivery mechanism for gene therapies. However, the expression profile of the receptor in cancer, particularly in clinical breast cancer, is poorly reported. This study evaluated the expression of CAR in human breast cancers. Frozen sections from breast cancer primary tumours (matched tumour n=114 and background n=30) were immunostained with CAR antibody. RNA was reverse transcribed and analysed by quantitative-PCR (Q-PCR). Levels of CAR were analysed against the clinical background, with a median follow-up of 72 months. Staining intensity of CAR was increased within tumour sections compared to background tissue. Q-PCR revealed significantly elevated levels of CAR transcript in breast tumours (32 313+/-7067 mean+/-SE vs. background tissue 11 483+/-7069, P=0.023). CAR expression also increased with grade of tumour. Patients who had tumour metastases also showed elevated levels of CAR expression (metastasis: 61 940+/-22 749 vs. alive and well 29 655+/-8149), however those with local recurrences had reduced levels of CAR. Ductal carcinomas expressed lower levels of CAR compared to tumours of other types. Tumours with nodal involvement were also associated with higher levels of CAR (node positive 2320 median vs. node negative 1077.5). Levels of CAR were significantly correlated with long-term survival over a period of 6 years (P=0.004, univariate analysis). We conclude that CAR expression is elevated in primary breast cancers. This may have a bearing on its use as means of delivery for gene therapy and suggests that further work is necessary to understand this complex molecule.

Comparison of the effects of risperidone and haloperidol on regional cerebral blood flow in schizophrenia.

BACKGROUND: Atypical antipsychotics, such as risperidone, have been shown to be more effective for the treatment of the symptoms of schizophrenia and have a greater beneficial effect on neurocognition compared to the conventional antipsychotics. The present study used [(15)O]H(2)O positron emission tomography imaging of regional cerebral blood flow to examine and compare the effects of haloperidol and risperidone on brain function. METHODS: Thirty-two subjects with schizophrenia participated in the study. Each subject was scanned in a medication-free state, and after being on a stable clinically assigned dose of either risperidone or haloperidol for 3 weeks. The off-medication scan was subtracted from the on-medication scan, using a within-subjects design. A randomization analysis was used to determine differences between the effects of haloperidol and risperidone on regional cerebral blood flow. RESULTS: Haloperidol was associated with a significantly greater increase in regional cerebral blood flow in the left putamen and posterior cingulate, and a significantly greater decrease in regional cerebral blood flow in frontal regions compared to risperidone. Risperidone was associated with a significantly greater decrease in regional cerebral blood flow in the cerebellum bilaterally compared to haloperidol. CONCLUSIONS: The results show that risperidone and haloperidol have significantly different effects on brain function, which may be related to their differences in efficacy and side effects. Further work is required to more precisely determine the mechanisms by which different antipsychotic medications exert their therapeutic effects on the clinical symptoms and cognition in schizophrenia. These findings emphasize the importance of controlling for both medication status and the individual antipsychotic in neuroimaging studies.

Effects of timing and duration of cognitive activation in [15O]water PET studies.

The multiple injection [15O]water method offers unique opportunities for studying cognitive processing by the human brain. The influence of the duration and temporal placement of an activation task, in relation to the arrival of the radiotracer in the brain, is a fundamental methodologic question for cognitive activation studies. A quantitative positron emission tomography (PET) study of five normal volunteers was performed in which the stimulation consisted of a visual activation task (alternating checkerboard pattern) superimposed on an auditory baseline task (syllable monitoring). Ten injection conditions, with varying duration and timing of the visual activation, were used. Regional CBF (rCBF) in visual cortex was measured quantitatively using the autoradiographic method. A 20-s stimulation, centered on the bolus arrival in the brain, produced significant changes in rCBF. Because varying the duration and timing of the activation task technically violates the temporal homogeneity assumption of the autoradiographic model, a mathematical simulation was formulated to evaluate the potential influence of these variations. Results of the simulation are consistent with the PET data and suggest that activation can be limited to a narrow temporal window centered on the radiotracer uptake. The ability to observe significant changes in rCBF with short stimulation intervals is of particular interest in the use of [15O]water PET for studies of cognitive processes with a short time course.

Sample size and statistical power in [15O]H2O studies of human cognition.

Determining the appropriate sample size is a crucial component of positron emission tomography (PET) studies. Power calculations, the traditional method for determining sample size, were developed for hypothesis-testing approaches to data analysis. This method for determining sample size is challenged by the complexities of PET data analysis: use of exploratory analysis strategies, search for multiple correlated nodes on interlinked networks, and analysis of large numbers of pixels that may have correlated values due to both anatomical and functional dependence. We examine the effects of variable sample size in a study of human memory, comparing large (n = 33), medium (n = 16,17), small (n = 11, 11, 11), and very small (n = 6,6,7,7,7) samples. Results from the large sample are assumed to be the "gold standard." The primary criterion for assessing sample size is replicability. This is evaluated using a hierarchically ordered group of parameters: pattern of peaks, location of peaks, number of peaks, size (volume) of peaks, and intensity of the associated t (or z) statistic. As sample size decreases, false negatives begin to appear, with some loss of pattern and peak detection; there is no corresponding increase in false positives. The results suggest that good replicability occurs with a sample size of 10-20 subjects in studies of human cognition that use paired subtraction comparisons of single experimental/baseline conditions with blood flow differences ranging from 4 to 13%.

Remembering the past: two facets of episodic memory explored with positron emission tomography.

OBJECTIVE: This study used positron emission tomography to examine two kinds of personal memory that are used in psychiatric evaluation: focused episodic memory (recall of past experience, employed in "taking a history") and random episodic memory (uncensored thinking about experience, examined during analytic therapy using free association). For comparison, a third memory task was used to tap impersonal memory that represents general information about the world ("semantic memory"). METHOD: Thirteen subjects were studied using the [15O]H2O method to obtain quantitative measurements of cerebral blood flow. The three conditions were subtracted and their relative relationships examined. RESULTS: The random episodic condition produced activations in widely distributed association cortex (right and left frontal, parietal, angular/supramarginal, and posterior inferior temporal regions). Focused episodic memory engaged a network that included the medial inferior frontal regions, precuneus/retrosplenial cingulate, anterior cingulate, thalamus, and cerebellum. The use of medial frontal regions and the precuneus/retrosplenial cingulate was common to both focused and random episodic memory. The major difference between semantic and episodic memory was activation of Broca's area and the left frontal operculum by semantic memory. CONCLUSIONS: These results indicate that free-ranging mental activity (random episodic memory) produces large activations in association cortex and may reflect both active retrieval of past experiences and planning of future experiences. Focused episodic memory shares some components of this circuit (inferior frontal and precuneus), which may reflect the time-linked components of both aspects of episodic memory, and which permit human beings to experience personal identity, consciousness, and self-awareness.