Mutant clones in normal epithelium outcompete and eliminate emerging tumours.

Human epithelial tissues accumulate cancer-driver mutations with age1-9, yet tumour formation remains rare. The positive selection of these mutations suggests that they alter the behaviour and fitness of proliferating cells10-12. Thus, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less competitive neighbours11-14. However, little is known about how such dynamic competition in normal epithelia influences early tumorigenesis. Here we show that the majority of newly formed oesophageal tumours are eliminated through competition with mutant clones in the adjacent normal epithelium. We followed the fate of nascent, microscopic, pre-malignant tumours in a mouse model of oesophageal carcinogenesis and found that most were rapidly lost with no indication of tumour cell death, decreased proliferation or an anti-tumour immune response. However, deep sequencing of ten-day-old and one-year-old tumours showed evidence of selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased early tumour removal, whereas pharmacological inhibition of clonal competition reduced tumour loss. These results support a model in which survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the surrounding normal tissue. Mutant clones in normal epithelium have an unexpected anti-tumorigenic role in purging early tumours through cell competition, thereby preserving tissue integrity.

Classifying suicidal behavior with resting-state functional connectivity and structural neuroimaging.

OBJECTIVE: About 80% of patients who commit suicide do not report suicidal ideation the last time they speak to their mental health provider, highlighting the need to identify biomarkers of suicidal behavior. Our goal is to identify suicidal behavior neural biomarkers to classify suicidal psychiatric inpatients. METHODS: Eighty percent of our sample [suicidal (n = 63) and non-suicidal psychiatric inpatients (n = 65)] was used to determine significant differences in structural and resting-state functional connectivity measures throughout the brain. These measures were used in a random forest classification model on 80% of the sample for training the model. RESULTS: The model built on 80% of the patients had sensitivity = 79.4% and specificity = 72.3%. This model was tested on an independent sample (20%; n = 32) with sensitivity = 81.3% and specificity = 75.0% for confirming the generalizability of the model. Altered resting-state functional connectivity features from frontal and middle temporal regions, as well as the amygdala, parahippocampus, putamen, and vermis were found to generalize best. CONCLUSION: This work demonstrates neuroimaging (an unbiased biomarker) can be used to classify suicidal behavior in psychiatric inpatients without observing any clinical features.

Insula and amygdala resting-state functional connectivity differentiate bipolar from unipolar depression.

OBJECTIVE: Distinguishing depressive episodes due to bipolar disorder (BD) or major depressive disorder (MDD) solely on clinical grounds is challenging. We aimed at comparing resting-state functional connectivity (rsFC) of regions subserving emotional regulation in similarly depressed BD and MDD. METHOD: We enrolled 76 in-patients (BD, n = 36; MDD, n = 40) and 40 healthy controls (HC). A seed-based approach was used to identify regions showing different rsFC with the insula and the amygdala. Insular and amygdalar parcellations were then performed along with diagnostic accuracy of the main findings. RESULTS: Lower rsFC between the left insula and the left mid-dorsolateral prefrontal cortex and between bilateral insula and right frontopolar prefrontal cortex (FPPFC) was observed in BD compared to MDD and HC. These results were driven by the dorsal anterior and posterior insula (PI). Lower rsFC between the right amygdala and the left anterior hippocampus was observed in MDD compared to BD and HC. These results were driven by the centromedial and laterobasal amygdala. Left PI/right FPPC rsFC showed 78% accuracy differentiating BD and MDD. CONCLUSION: rsFC of amygdala and insula distinguished between depressed BD and MDD. The observed differences suggest the possibility of differential pathophysiological mechanisms of emotional dysfunction in bipolar and unipolar depression.

A qualitative and quantitative assessment of the impact of three processing algorithms with halving of study count statistics in myocardial perfusion imaging: filtered backprojection, maximal likelihood expectation maximisation and ordered subset expectation maximisation with resolution recovery.

INTRODUCTION: Ordered subset expectation maximisation with depth-dependent resolution recovery (OSEM-RR) is a processing algorithm reported to improve images with halved tracer activity in myocardial perfusion scintigraphy (MPS) compared to filtered backprojection (FBP) using conventional activities. OSEM-RR has not yet been compared with maximal likelihood expectation maximisation (MLEM). METHODS: 39 patients undergoing MPS and two anthropomorphic phantoms (one with, one without an inferior wall insert) had full-time (FT) and half-time (HT) SPECT datasets acquired simultaneously and processed by FBP, MLEM and OSEM-RR. Two experienced reporters scored images of all clinical studies (n=234) for conspicuity of a perfusion defect, with results being compared using Wilcoxon paired and Kappa tests. A quantitative assessment based on mean segmental pixel counts taken from numbers automatically displayed over the 20 segments of Cedars Sinai Autoquant QPS image were compared using Pearson's correlation and Bland Altman analysis. RESULTS: A small but consistent superior concurrence between FT and HT datasets for OSEM-RR compared to FBP and MLEM was observed for both qualitative and quantitative analyses. OSEM-RR resulted in better definition of the inferior wall defect on the phantom study. CONCLUSION: OSEM-RR appears superior to both FBP and MLEM in terms of handling reduced count statistics.

The gut microbiota is associated with psychiatric symptom severity and treatment outcome among individuals with serious mental illness.

BACKGROUND: Emerging evidence implicates the gut microbiota in central nervous system functioning via its effects on inflammation, the hypothalamic-pituitary axis, and/or neurotransmission. Our understanding of the cellular underpinnings of the brain-gut relationship is based almost exclusively on animal models with some small-scale human studies. This study examined the relationship between the gut microbiota and psychiatric symptom severity and treatment response among inpatients with serious mental illness. METHOD: We collected data from adult inpatients (N = 111). Measures of diagnoses, suicide severity, trauma, depression, and anxiety were collected shortly after admission, while self-collected fecal swabs were collected early in the course of hospitalization and processed using 16S rRNA gene sequencing and whole genome shotgun sequencing methods. RESULTS: Results indicate that depression and anxiety severity shortly after admission were negatively associated with bacterial richness and alpha diversity. Additional analyses revealed a number of bacterial taxa associated with depression and anxiety severity. Gut microbiota richness and alpha diversity early in the course of hospitalization was a significant predictor of depression remission at discharge. CONCLUSIONS: This study is among the first to demonstrate a gut microbiota relationship with symptom severity among psychiatric inpatients as well as a relationship to remission of depression post-treatment. These findings are consistent with animal models and limited human studies as well as with the broader literature implicating inflammation in the pathophysiology of depression. These findings offer the foundation for further studies of novel therapeutic approaches to the treatment, prevention of, or recurrence of serious mental illness.

Contributions of Na+ flux and the anoxic depolarization to adenosine 5'-triphosphate levels in hypoxic/hypoglycemic rat hippocampal slices.

A 10 min exposure of rat hippocampal slices to hypoxic/hypoglycemic medium decreased tissue adenosine 5'-triphosphate (ATP) levels. Hypoxia/hypoglycemia also caused an anoxic depolarization and essentially no recovery of the synaptically evoked population spike from CA1 region recorded 30 min after re-introduction of normoxic/normoglycemic medium. Removal of Ca2+ or the addition of either the non-competitive N-methyl-D-aspartate antagonist dizocilpine maleate, the inorganic Ca2+ channel antagonist Co2+; or the Na+ channel blocker tetrodotoxin to hypoxic/hypoglycemic medium improved recovery of the evoked population spike upon re-oxygenation. Dizocilpine maleate, Co2+, and tetrodotoxin spared ATP during exposure to hypoxia/hypoglycemia. In contrast, Ca(2+)-free medium facilitated recovery of the population spike but did not preserve ATP during hypoxia/hypoglycemia. Dizocilpine maleate, Co2+ or dantrolene, when added to Ca(2+)-free medium, did not preserve ATP. Tetrodotoxin, when added to Ca(2+)-free medium, was effective in sparing ATP in hypoxic/hypoglycemic medium. To determine the effect of anoxic depolarization on ATP levels, hippocampal slices were collected just before and after the depolarization. There appeared to be an abrupt drop in ATP associated with the anoxic depolarization. We conclude that Na+ influx plays a relatively larger role in ATP consumption during hypoxia/hypoglycemia than Ca2+ influx. In addition, the anoxic depolarization imposes a large and rapid drop in ATP levels.

Analysis of mixed human/microbial DNA samples: a validation study of two PCR AMP-FLP typing methods.

The reliability of the PCR technique used to type two human variable number tandem repeats, that is, 3' to apolipoprotein B gene and locus D17S30, was examined using DNA samples of mixed human and microbial origin. Mixtures of human and microbial DNA were amplified, choosing microbes found commonly in the vagina. Total inhibition of human amplification and/or "drop-out" of the larger amplification fragment length polymorphism allele was observed at both loci in the presence of DNA from some vaginal micro-flora.

Enrichment of microsatellites from the citrus genome using biotinylated oligonucleotide sequences bound to streptavidin-coated magnetic particles.

A method is described for the rapid isolation of microsatellite sequences using a biotin-labeled oligonucleotide attached to streptavidin-coated magnetic particles. The oligonucleotide "hook" in solution hybridizes to complementary single-stranded lengths of genomic DNA onto which have been engineered specific PCR priming sites. The final product is an enriched library of microsatellites of defined sequences. The method is applicable to any genome and in principle is adaptable to the rapid isolation of both repetitive as well as genic sequences. It is illustrated by the isolation of trinucleotide repeat (TAA)n sequences from the citrus genome.

Phorbol ester alters rat hippocampal neuronal response to hypoxia.

The effect of the protein kinase C (PKC) activator, 4 beta-phorbol-12, 13-dibutyrate (beta-PDBu) on electrophysiological properties of rat hippocampal CA1 neurons exposed to moderate hypoxia was examined. Hypoxic beta-PDBu-pretreated neurons differed from untreated neurons by exhibiting an adenosine-independent loss of synaptic transmission, an apparent large increase in injected threshold current necessary to elicit an action potential, an increase rather than a decrease in membrane resistance, and an increase rather than a decrease in hyperpolarizing holding current. Additional experiments with an adenosine A1 antagonist suggest that beta-PDBu alters neuronal responses to hypoxia through some mechanism other than the documented ability of beta-PDBu to uncouple the A1 receptor.

Modulation of neuronal excitability by endogenous adenosine in the absence of synaptic transmission.

Rat hippocampal slices were superfused with low calcium, high magnesium medium. Reductions in flow rate were associated with a marked depression of antidromically elicited afterpotentials with little change in the initial antidromic population spike recorded from CA1 pyramidal neurons. The depression of the afterpotential at the lower flow rates was largely reversed by the adenosine antagonist, theophylline (100 microM), by adenosine deaminase (10 micrograms/ml) and was mimicked by the application of the adenosine reuptake blocker, dipyridamole (100 microM). Since synaptic transmission was blocked, it is concluded that sufficient endogenous adenosine exists in the absence of synaptic function to alter neuronal excitability.

Adenosine antagonists alter the synaptic response to in vitro ischemia in the rat hippocampus.

Exposure of the submerged hippocampal slice to in vitro ischemic conditions (superfusion with hypoxic medium lacking glucose) resulted in a progression of changes in the orthodromically evoked response recorded from the CA1 pyramidal region. There was an early depression of the population spike with no change in the presynaptic fiber volley, followed by a transient return of the population spike and, finally, a complete loss of both the population spike and fiber volley. The adenosine A1 subtype-selective antagonists, 8-phenyltheophylline (8-PT) and 8-cyclopentyltheophylline (8-CPT), greatly attenuated the early depression of the population spike such that the initial loss of the population spike was associated with the loss of the fiber volley. This result suggests that the initial loss of synaptic function in the hippocampal slice during exposure to in vitro ischemic conditions is due to increased levels of the inhibitory neuromodulator, adenosine.

Adenosine antagonists delay hypoxia-induced depression of neuronal activity in hippocampal brain slice.

Submerged rat hippocampal slices were exposed to hypoxic medium prepared with 95% N2/5% CO2. The population spikes recorded from CA1 cell layer were completely blocked within a range of 5-10 min. The adenosine antagonist theophylline (100 microM) delayed and partially prevented the hypoxia-induced depression. Increasing concentrations of the more potent adenosine antagonist 8-phenyltheophylline (8-PT; 0.1, 1, 10 microM) resulted in progressively less hypoxia-induced depression. The antidromically elicited afterpotentials recorded in the absence of synaptic transmission in low calcium, high magnesium medium were blocked within 8 min of hypoxia. Theophylline (100 microM) and 8-PT (10 microM) delayed to a similar extent the hypoxia-induced depression of the first afterpotential but did not prevent its complete depression.

Escape from inhibition of synaptic transmission during in vitro hypoxia and hypoglycemia in the hippocampus.

Electrophysiological recordings were made from rat hippocampal slices exposed to in vitro ischemic conditions in which the superfused medium is hypoxic and lacking glucose. Under these conditions, the evoked population spike recorded in CA1 is initially depressed and then transiently returns prior to an anoxic depolarization. This transient return in synaptic function under ischemic-like conditions also occurs if the population spike is inhibited by pretreatment with adenosinergic agonists or with the gamma-aminobutyric acid (GABA)B agonist, baclofen.

Hydrogen peroxide opposes the hypoxic depression of evoked synaptic transmission in rat hippocampal slices.

Hydrogen peroxide (H2O2, 3.3 mM) partially reversed the hypoxic depression of the evoked population spike recorded from CA1 region of rat hippocampal slices. It is known that elevated endogenous adenosine contributes to the hypoxic inhibition of the population spike. Exogenous adenosine (100 microM) inhibited the population spike that had been partially resuscitated by H2O2 during maintained hypoxia. It is concluded that the ability of H2O2 to oppose hypoxic depression does not occur at the level of the adenosine receptor since added adenosine was still effective in inhibiting the evoked potential in the presence of H2O2.

The effect of pentylenetetrazol on inward currents of non-bursting neurons and its role in plateau formation.

The epileptogenic drug, pentylenetetrazol (PTZ) produces paroxysmal depolarization shifts in molluscan neurons that are similar to PDSs seen at a mammalian epileptic focus. Most research on molluscan neurons indicates that PTZ acts by altering ionic somatic conductances. This study was carried out to investigate the effect of PTZ on inward currents in isolated neurons of the pond snail, Lymnaea stagnalis, and to investigate how these altered currents might lead to the production of PDSs. In concentrations from 10 to 60 mM, PTZ decreased maximum inward current conductance and shifted the inactivation and activation curves to the left with the former shift being consistently greater. There was no change in reversal potential or time constants for activation and inactivation of inward currents. The effects of the PTZ-induced alterations in the inward currents were studied by incorporating them along with alterations of outward currents seen in this and other studies in a computer model for molluscan neuronal firing. The composite model reproduced in large part the intermediate changes in electrical activity seen before the development of the PDS as well as the PDS.

Prostaglandins block a Ca2+-dependent slow spike afterhyperpolarization independent of effects on Ca2+ influx in visceral afferent neurons.

The blockade of a slow Ca2+-activated K+-dependent afterhyperpolarization (AHPs) in rabbit visceral sensory neurons by the prostaglandins, PGE1 and PGD2, was investigated to determine whether the blockade was indirectly due to a reduction in Ca2+ influx. The prostaglandins (PGs) could block the AHPs in the absence of any change in Ca2+-dependent spikes elicited in the presence of tetrodotoxin and tetraethylammonium bromide. A PG-induced decrease in Ca2+-dependent spike width observed in some neurons was temporally dissociated from the PG-induced block of the AHPs. In addition, a slow afterhyperpolarization produced by the application of the Ca2+ ionophore, A23187, was blocked by the PGs. It is concluded that a reduction in Ca2+ influx is not responsible for the PG-induced blockade of the AHPs.

Hydroxylamine blocks pre- but not postsynaptic adenosine A(1) receptor-mediated actions in rat hippocampus.

The commonly used nitric oxide donor, hydroxylamine (NH(2)OH), can block or reverse the inhibition of glutamatergic transmission by adenosine or an adenosine A(1) agonist in rat hippocampal slice. In these experiments, hydroxylamine did not affect the adenosine A(1) receptor-mediated depression of postsynaptic excitability. We conclude that hydroxylamine acts presynaptically to counter adenosine A(1) receptor-mediated inhibition of synaptic transmission.

Hydroxylamine blocks adenosine A1 receptor-mediated inhibition of synaptic transmission in rat hippocampus.

The nitric oxide donor hydroxylamine (NH2OH) induced a transient depression of the evoked synaptic potential recorded in the rat hippocampal CA1 region. This depression was abolished with an adenosine A1 antagonist, 8-cyclopentyltheophylline. In addition, hydroxylamine reversed adenosine A1 receptor-mediated inhibition of the evoked population spike, the fEPSP and the intracellularly recorded EPSP. The inhibitory modulation of adenosine A1 receptor activation by hydroxylamine suggests the presence of a potent endogenous regulatory site.

Antagonism of the responses to isoproterenol in the rat hippocampal slice with subtype-selective antagonists.

The electrophysiological and cAMP responses to the beta-adrenoceptor agonist isoproterenol were measured in the in vitro hippocampal slice preparation. Subtype-selective antagonists were used to evaluate the specificity of these responses. The beta 1-selective antagonist ICI 89,406 was 60-fold more potent than was the beta 2-selective antagonist ICI 118,551 at antagonizing the electrophysiological response. ICI 89,406 was 200 times more potent in its antagonism of the cAMP response. These results suggest that the electrophysiological and cAMP responses in this preparation are primarily mediated by beta 1-adrenoceptors.

Purine release and inhibition of synaptic transmission during hypoxia and hypoglycemia in rat hippocampal slices.

Evoked synaptic potentials and purine efflux were measured simultaneously from rat hippocampal slices. Slices were exposed to hypoxia, to glucose-free medium, and to in vitro ischemia consisting of glucose-free, hypoxic medium. During exposure to hypoxia or the glucose-free condition, radiolabelled purine efflux increased and the evoked population spike declined. Synaptic potentials and purine efflux returned to baseline values after reintroduction of normoxic and normoglycemic medium. During exposure to in vitro ischemia, purine and adenosine efflux were greatly increased with the appearance of the anoxic depolarization.