Subtype-specific circadian clock dysregulation modulates breast cancer biology, invasiveness, and prognosis.

Studies in shift workers and model organisms link circadian disruption to breast cancer. However, molecular rhythms in non-cancerous and cancerous human breast tissues are largely unknown. We reconstructed rhythms informatically, integrating locally collected, time-stamped biopsies with public datasets. For non-cancerous tissue, the inferred order of core-circadian genes matches established physiology. Inflammatory, epithelial-mesenchymal transition (EMT), and estrogen responsiveness pathways show circadian modulation. Among tumors, clock correlation analysis demonstrates subtype-specific changes in circadian organization. Luminal A organoids and informatic ordering of Luminal A samples exhibit continued, albeit disrupted rhythms. However, CYCLOPS magnitude, a measure of global rhythm strength, varied widely among Luminal A samples. Cycling of EMT pathway genes was markedly increased in high-magnitude Luminal A tumors. Patients with high-magnitude tumors had reduced 5-year survival. Correspondingly, 3D Luminal A cultures show reduced invasion following molecular clock disruption. This study links subtype-specific circadian disruption in breast cancer to EMT, metastatic potential, and prognosis.

Creation and characterization of free-standing cryogenic targets for laser-driven ion acceleration.

A technique for the creation of free-standing cryogenic targets for laser-driven ion acceleration is presented, which allows us to create solid state targets consisting of initially gaseous materials. In particular, the use of deuterium and the methods for its preparation as a target material for laser-driven ion acceleration are discussed. Moving in the phase diagram through the liquid phase leads to the substance covering an aperture on a cooled copper frame where it is solidified through further cooling. An account of characterization techniques for target thickness is given, with a focus on deducing thickness values from distance values delivered by chromatic confocal sensors.

Caecum herniation through the Foramen of Winslow.

Foramen of Winslow herniation is a rare occurrence with a high mortality; it presents a diagnostic challenge with subtle clinical and radiological features. We present a case of caecal herniation through the foramen of Winslow creating a closed loop obstruction which remained undiagnosed until laparotomy. Reduction was achieved with gentle traction after first decompressing the caecum whilst still within the lesser sac.

Identification and functional analysis of SKA2 interaction with the glucocorticoid receptor.

Glucocorticoid (GC) receptors (GRs) have profound anti-survival effects on human small cell lung cancer (SCLC). To explore the basis of these effects, protein partners for GRs were sought using a yeast two-hybrid screen. We discovered a novel gene, FAM33A, subsequently identified as a SKA1 partner and involved in mitosis, and so renamed Ska2. We produced an anti-peptide antibody that specifically recognized full-length human SKA2 to measure expression in human cell lines and tissues. There was a wide variation in expression across multiple cell lines, but none was detected in the liver cell line HepG2. A xenograft model of human SCLC had intense staining and archival tissue revealed SKA2 in several human lung and breast tumours. SKA2 was found in the cytoplasm, where it co-localized with GR, but nuclear expression of SKA2 was seen in breast tumours. SKA2 overexpression increased GC transactivation in HepG2 cells while SKA2 knockdown in A549 human lung epithelial cells decreased transactivation and prevented dexamethasone inhibition of proliferation. GC treatment decreased SKA2 protein levels in A549 cells, as did Staurosporine, phorbol ester and trichostatin A; all agents that inhibit cell proliferation. Overexpression of SKA2 potentiated the proliferative response to IGF-I exposure, and knockdown with shRNA caused cells to arrest in mitosis. SKA2 has recently been identified in HeLa S3 cells as part of a complex, which is critical for spindle checkpoint silencing and exit from mitosis. Our new data show involvement in cell proliferation and GC signalling, with implications for understanding how GCs impact on cell fate.

Site-specific, inflammation-induced adaptations in withdrawal reflex pathways in the anesthetized rabbit.

Transmission in spinal reflex pathways from the toes to the ankle flexor tibialis anterior (TA) and to the knee flexor semitendinosus (ST), and from the heel to the ankle extensor medial gastrocnemius (MG), has been studied during antigen-induced inflammation due to subcutaneous injection of ovalbumin at the heel or at the toes in pre-immunized, pentobarbitone-anesthetized rabbits. The ovalbumin challenge induced a mild swelling at the injection site that developed over 6 h. Inflammation at the toes facilitated both flexor reflexes evoked from the toes and inhibited MG extensor responses to stimulation at the heel, with effects usually developing within 75 min of the initial injection and persisting to the end of the 6 h post-injection recording period. When inflammation was induced at the heel, the heel-MG and toes-ST reflexes were enhanced, whereas the toes-TA reflex was inhibited. The findings support the view that long-lasting changes in reflexes evoked by noxious stimuli have evolved to enhance reflex protection of an injured site.

Aberrant activation of notch signaling in human breast cancer.

A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jkappa-dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease.

The organization of motor responses to noxious stimuli.

Withdrawal reflexes are the simplest centrally organized responses to painful stimuli, making them popular models for the study of nociception. Until recently, it was believed that withdrawal was a single reflex response involving excitation of all flexor muscles in a limb with concomitant inhibition of extensors. However, recent findings suggest that withdrawal reflexes are tailored to produce the most appropriate movement according the site at which the stimulus is applied, which could require extensors to act as the primary movers. This idea is supported by new evidence obtained from the direct measurement of limb movements, although these data indicate that differentiation of withdrawal reflexes is most readily seen from stimuli applied to the plantar surface of the foot. Injurious stimuli augment the protective function of reflexes by enhancing (sensitizing) reflexes that protect the injured site and inhibiting those reflexes that might exacerbate the insult. The areas from which a reflex can be sensitized closely match those from which the reflex itself can be evoked, provided that the spinal cord is intact. If descending pathways are interrupted, sensitization can be evoked from a much wider area. Thus, the exact movement made in a withdrawal reflex is determined by the location of the evoking stimulus and whether the reflex sensitized or inhibited after an injury depends on the relationship between the site of the injury and the movement made by the reflex. The factors should be borne in mind when designing experiments in which reflexes are used as the end point in studies of nociception.

Differential modulation of withdrawal reflexes by a cannabinoid in the rabbit.

Inhibition of spinal and trigeminal withdrawal reflexes by morphine and by the cannabinoid agonist HU 210 has been studied in anaesthetized and in decerebrated rabbits. In intact, pentobarbitone-anaesthetized animals, the jaw-depressor reflex (JDR) evoked by stimulation of the tongue, and the reflex elicited in the ankle flexor tibialis anterior (TA) by stimulation of the toes were inhibited to the same extent by morphine (1-30 mg kg(-1) i.v. cumulative). In spinalized, anaesthetized rabbits morphine depressed the JDR to the same level as in non-spinal preparations, but the effect of the opioid on the TA reflex was significantly reduced. All effects of morphine were reversed by naloxone (0.25 mg kg(-1), i.v.). In anaesthetised intact animals, HU 210 depressed the JDR at a dose of 100 nmol kg(-1) i.v. cumulative, reduced reflexes evoked in the knee flexor muscle semitendinosus (ST) by stimulation at the toes at a dose of 30 nmol kg(-1) i.v. cumulative, but had no consistent or significant effects on the TA reflex to toe stimulation. The same results were obtained in spinalized, anaesthetised animals. In decerebrated, spinalized rabbits with no anaesthesia, HU 210 (30 nmol kg(-1)) depressed both ST and TA reflexes evoked by toe stimulation. These data reveal that trigeminal and spinal withdrawal reflexes are equally sensitive to morphine provided the spinal cord is intact, suggesting that at least part of the action of systemic morphine is due to activation of descending inhibition. The present results also show for the first time that cannabinoid agonists can inhibit trigeminal withdrawal reflexes. HU 210 had differential effects on the three reflexes studied depending on the presence or absence of anaesthesia. This is the first occasion on which we have found pharmacological distinctions between withdrawal reflexes, and indicates that spinal sensorimotor processing is more heterogeneous than has been suspected previously.

5-HT receptors involved in opioid-activated descending inhibition of spinal withdrawal reflexes in the decerebrated rabbit.

The role of 5-HT(1B/1D), 5-HT(2) and 5-HT(3) receptors in mediating descending inhibition of spinal reflexes activated by application of fentanyl to the fourth ventricle has been studied in rabbits decerebrated under N(2)O/isoflurane anaesthesia. In the control state, intraventricular fentanyl (3-30 microg kg(-1)) depressed, to an equal extent, short- and long-latency reflexes in the medial gastrocnemius muscle nerve evoked by electrical stimulation of all sural nerve afferents. Inhibition of reflexes resulted from a decreased base line excitability in the reflex pathway accompanied by a reduction in the rate of temporal summation of responses. Fentanyl-induced suppression of short- and long-latency reflexes was significantly reduced after intrathecal administration of the selective 5-HT(2)-receptor antagonist ICI 170,809 (300 microg). The same dose of the selective 5-HT(1B/1D) blocker GR 127,935 reduced inhibition from intraventricular fentanyl only for long-latency reflexes (i.e. those parts of the response for which the afferent drive is provided mainly by Adelta and C-fibre afferents). The 5-HT(3) antagonist tropisetron (also 300 microg intrathecal) did not significantly alter the descending inhibition of reflexes evoked by fentanyl. Both GR 127,935 and tropisetron reduced temporal summation of reflexes per se, effects that were reversed by intraventricular fentanyl. These data suggest that the descending pathway(s) activated by intraventricular fentanyl liberate 5-HT in the spinal cord to inhibit withdrawal reflexes by acting at 5-HT(2) and 5-HT(1B/1D), but not 5-HT(3) receptors. 5-HT(1B/1D), and to a lesser extent 5-HT(3) receptors also appear to have a role in modulating temporal summation of reflexes evoked by repetitive stimuli.

Endogenous opioids support the spinal inhibitory action of an alpha 2-adrenoceptor agonist in the decerebrated, spinalised rabbit.

The present study examined the possible contribution of endogenous opioids to inhibition of spinal reflexes by an alpha(2)-adrenoceptor agonist. In rabbits decerebrated and spinalised under halothane/nitrous oxide anaesthesia, the selective alpha(2)-adrenoceptor agonist dexmedetomidine (3-30 microg intrathecal) induced significant decreases in short- and long-latency reflex responses evoked in medial gastrocnemius (MG) motoneurones by stimulation of the sural nerve. After recovery from dexmedetomidine, the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA; 100 microg intrathecal) significantly enhanced short-latency but not long-latency MG reflex responses. After beta-FNA, inhibition of all reflexes by dexmedetomidine was significantly weaker than in the control state, whereas the cardiovascular actions of dexmedetomidine were unaffected. These data confirm that activation of spinal alpha(2)-adrenoceptors depresses MG reflexes evoked by all groups of sural nerve afferent fibres, and shows that endogenous opioid tone supports the inhibitory action of alpha(2) agonists, possibly by a synergistic interaction in the spinal cord.

Organisation of sensitisation of hind limb withdrawal reflexes from acute noxious stimuli in the rabbit.

Spatial aspects of central sensitisation were investigated by studying the effects on three hind limb withdrawal reflexes of an acute noxious stimulus (20 % mustard oil) applied to a number of locations around the body in decerebrate and in anaesthetised rabbits. Reflex responses to electrical stimulation of the toes were recorded from the ankle flexor tibialis anterior (TA) and the knee flexor semitendinosus (ST), whereas responses to stimulation of the heel were recorded from the ankle extensor medial gastrocnemius (MG). In non-spinalised, decerebrated, pentobarbitone-sedated preparations, flexor reflexes were facilitated significantly from sites on the plantar surface of the ipsilateral foot but were either inhibited or unaffected by stimulation of sites away from this location. The heel-MG reflex was facilitated from the ipsilateral heel and was inhibited from a number of ipsilateral, contralateral and off-limb sites. In decerebrated, spinalised, pentobarbitone-sedated animals, mustard oil applied to any site on the ipsilateral hind limb enhanced both flexor reflexes, whereas the MG reflex was enhanced only after stimulation at the ipsilateral heel and was inhibited after stimulation of the toe tips or TA muscle. Mustard oil on the contralateral limb had no effect on any reflex. In rabbits anaesthetised with pentobarbitone and prepared with minimal surgical interference, the sensitisation fields for the heel-MG and toes-TA reflexes were very similar to those in non-spinal decerebrates whereas that for toes-ST was more like the pattern observed in spinalised animals. In no preparation was sensitisation or inhibition of reflexes related to the degree of motoneurone activity generated in direct response to the sensitising stimulus. This study provides for the first time a complete description of the sensitisation fields for reflexes to individual muscles. Descending controls had a marked effect on the area from which sensitisation of flexor reflexes could be obtained, as the sensitisation fields for the flexor reflexes evoked from the toes were larger in spinalised compared to decerebrated, non-spinalised animals. The intermediate sizes of sensitisation fields in anaesthetised animals suggests that the area of these fields can be dynamically controlled from the brain. On the other hand, the sensitisation field for the heel-MG reflex varied little between preparations and appears to be a function of spinal neurones.

The involvement of bulbospinal pathways in fentanyl-induced inhibition of spinal withdrawal reflexes in the decerebrated rabbit.

The selective opioid OP3(mu)-receptor agonist fentanyl was administered via the intravenous, intrathecal and intraventricular routes to decerebrated rabbits in doses from 1-30 microg/kg. Reflexes evoked in medial gastrocnemius motoneurones by electrical stimulation of the sural nerve were depressed by fentanyl given by all three routes. The opioid was most potent when given intrathecally and least potent when given into the fourth ventricle. Blockade of spinal alpha2-adrenoceptors by intrathecal RX 821002 (100 microg) reduced the effectiveness of intrathecal and low (<3 microg/kg) intravenous doses of fentanyl, but did not affect or enhanced responses to high intraventricular and intravenous doses. Spinalization reduced the effectiveness of intrathecal and intravenous fentanyl and abolished inhibition from intraventricular dosing. These data show that fentanyl acts in the spinal cord and in the brain stem to suppress spinal reflexes, although very high doses were required for effects from the latter site. It appears that low intravenous doses of fentanyl act mainly in the spinal cord and that increasing the dosage recruits descending inhibition. The results of alpha2-adrenoceptor blockade indicate that the spinal inhibitory effects of opioids are enhanced by an interaction with endogenous noradrenaline in the spinal cord. Thus, the full expression of the spinal inhibitory effects of fentanyl is dependent on brain stem cell groups, either as a source of noradrenaline input to the spinal cord, or as a site from which opioids can activate descending inhibitory systems.