MicroRNAs as biomarkers for major depression: a role for let-7b and let-7c.

There is a growing emphasis in the field of psychiatry on the need to identify candidate biomarkers to aid in diagnosis and clinical management of depression, particularly with respect to predicting response to specific therapeutic strategies. MicroRNAs are small nucleotide sequences with the ability to regulate gene expression at the transcriptomic level and emerging evidence from a range of studies has highlighted their biomarker potential. Here we compared healthy controls (n=20) with patients diagnosed with major depression (n=40) and who were treatment-resistant to identify peripheral microRNA biomarkers, which could be used for diagnosis and to predict response to electroconvulsive therapy (ECT) and ketamine (KET) infusions, treatments that have previously shown to be effective in treatment-resistant depression (TRD). At baseline and after treatment, blood samples were taken and symptom severity scores rated using the Hamilton Depression Rating Scale (HDRS). Samples were analyzed for microRNA expression using microarray and validated using quantitative PCR. As expected, both treatments reduced HDRS scores. Compared with controls, the baseline expression of the microRNA let-7b was less by ~40% in TRD patients compared with controls. The baseline expression of let-7c was also lower by ~50% in TRD patients who received ECT. Bioinformatic analysis revealed that let-7b and let-7c regulates the expression of 27 genes in the PI3k-Akt-mTOR signaling pathway, which has previously been reported to be dysfunctional in depression. The expression of miR-16, miR-182, miR-451 and miR-223 were similar to that in controls. Baseline microRNA expression could not predict treatment response and microRNAs were unaffected by treatment. Taken together, we have identified let-7b and let-7c as candidate biomarkers of major depression.

Acidosis in the hospital setting: is metformin a common precipitant?

BACKGROUND: Acidosis is commonly seen in the acute hospital setting, and carries a high mortality. Metformin has been associated with lactic acidosis, but it is unclear how frequently this is a cause of acidosis in hospitalized inpatients. The aim of this study is to explore the underlying comorbidities and acute precipitants of acidosis in the hospital setting, including the relationship between type 2 diabetes (T2DM) and metformin use. METHODS: Retrospective review. Cases of acidosis were identified using the hospital discharge code for acidosis for a 3-month period: October-December 2005. RESULTS: A total of 101 episodes of acidosis were identified: 29% had isolated respiratory acidosis, 31% had metabolic acidosis and 40% had a mixed respiratory and metabolic acidosis. There were 28 cases of confirmed lactic acidosis. Twenty-nine patients had T2DM, but only five of the subjects with T2DM had lactic acidosis; two were on metformin. The major risk factors for development of lactic acidosis were hepatic impairment (OR 33.8, P = 0.01), severe left ventricular dysfunction (OR 25.3, P = 0.074) and impaired renal function (OR 9.7, P = 0.09), but not metformin use. CONCLUSION: Most cases of metabolic and lactic acidosis in the hospital setting occur in patients not taking metformin. Hepatic, renal and cardiac dysfunction are more important predictors for the development of acidosis.

Characterization of the feeding inhibition and neural activation produced by dorsomedial hypothalamic cholecystokinin administration.

Within the dorsomedial hypothalamus (DMH), cholecystokinin (CCK) has been proposed to modulate neuropeptide Y (NPY) signaling to affect food intake. However, the neural circuitry underlying the actions of this CCK-NPY signaling system in the controls of food intake has yet to be determined. We sought to characterize the feeding inhibition and brain neural activation produced by CCK administration into the DMH of rats. We determined the time course of feeding inhibitory effects of exogenous DMH CCK, assessed NPY gene expression in the DMH in response to DMH CCK administration, and characterized c-Fos activation in the entire brain induced by CCK injection into the DMH using c-Fos like immunohistochemistry. We found that parenchymal injection of CCK into the DMH decreased food intake during the entire 22 h observation period, with a primary effect in the first 4 h, and down-regulated NPY gene expression in the DMH. c-Fos immunohistochemistry revealed that DMH CCK increased the number of c-Fos positive cells in the paraventricular nucleus (PVN), arcuate nucleus, suprachiasmatic nucleus and retrochiasmatic area as well as in the contralateral DMH. This pattern of activity is different from that produced by peripherally administered CCK which is short acting and primarily activates neurons in the nucleus of the solitary tract and area postrema, as well as the PVN and DMH. Together, these data suggest that DMH CCK plays an important role in the control of food intake, and does so by activating different pathways from those activated by peripheral CCK.

Postnatal behavioral and physiological responses of piglets from gilts housed individually or in groups during gestation.

Gestational housing of sows remains a controversial issue that may affect the well-being of both sows and piglets. Therefore, 2 types of gestational housing were used to evaluate the stress imposed on pregnant gilts by each system and the effects on the offspring by comparing production, physiology, and behavioral measures of the piglets. Forty-eight Landrace x Yorkshire gilts were randomly assigned to groups (G) of 4 per pen (n = 8 pens; 3.9 m x 2.4 m) or to individual stalls (S; n = 16 stalls; 2.21 m x 0.61 m). Gilts were moved into individual farrowing crates 5 d before the expected farrowing date. Piglets were weighed at birth, d 14, and d 35. Two barrows from each litter were weaned at d 14 (early weaning) and housed together in pens. Maintenance behaviors (head in feeder, drinking, lying, eating mash) were videotaped and observed for the first 3 d after weaning using a 10-min interval scan sampling. Belly nosing and play/fight interactions were recorded from video observations for 3 d postweaning. An isolation test (30-min duration) was performed on one piglet from each pen of barrows on d 35. Time spent lying, the number of jumps against test box walls, and grunts and squeals were recorded in real time. Salivary cortisol was collected at 30-min intervals from baseline, and 0, 30, 60, and 90 min posttest. Jugular blood was collected from 2 barrows from each litter on d 1, 7, 14, 17, 21, and 28. Plasma TNF-alpha was analyzed by ELISA, and haptoglobin, alpha1-acid glycoprotein, and immunoglobulin G were analyzed by radial immunodiffusion. More piglets from the S treatment needed to be fed a liquid feed at weaning and drank more frequently on d 2 postweaning (P < 0.05). Additionally, by d 35 piglets from S gilts had a lighter BW (10.3 kg) than G piglets (12.8 kg; P < 0.01). Piglets from S gilts also grunted more during the 30-min isolation test (number of grunts = 356) than G piglets (number of grunts = 138; P < 0.01). Salivary cortisol and immune measures were not different. These data show some behavioral and production differences between piglets from individually stalled gilts and group-housed gilts. Therefore, there may be production advantages to housing first parity gilts in groups.

Neural Probes for Concurrent Detection of Neurochemical and Electrophysiological Signals in vivo.

Electrochemical sensing with microelectrode arrays provides a means for monitoring neurotransmitter dynamics across multiple locations within a micro-scale region of brain tissue. Here we present a multi-modal neural probe design for concurrent recording of neurochemical and electrophysiological signals in vivo. Prior to implantation, platinum sites on each array underwent platinum-black electroplating and Nafion electropolymerization, which increased sensitivity to dopamine by 74% and decreased sensitivity to common interferents by at least 89%. In a series of three rats, we applied various electrochemical waveforms to platinum sites and monitored neural activity on adjacent iridium sites. We found that chronoamperometry and constant-potential amperometry did not alter firing rates at +0.25, +0.50, and +0.75 V. In addition, we have demonstrated multi-modal recordings of striatal neurons in response to medial forebrain bundle stimulation.

Exploiting changes in the tumour microenvironment with sequential cytokine and matrix metalloprotease inhibitor treatment in a murine breast cancer model.

The study of treatment-induced changes in the tumour microenvironment might lead to effective combinations of biological therapy. IL-12 induced tumour regression and cure of an experimental murine breast cancer, HTH-K, but only after long-term treatment that was associated with chronic toxicity. During IL-12 therapy, tumour levels of the matrix metalloprotease MMP-9 declined and its inhibitor TIMP-1 was strongly induced. We therefore administered alternate cycles of IL-12 and the MMP inhibitor Batimastat (BB94) to mice. Therapeutic efficacy was increased compared with short-term IL-12 therapy but without the chronic toxicity associated with long-term IL-12 treatment. Image analysis of treated tumours revealed that BB94 prevented regeneration of tumour and stromal compartments that normally occurred after short-term IL-12 therapy.

A matrix metalloproteinase inhibitor which prevents fibroblast-mediated collagen lattice contraction.

Matrix metalloproteinases (MMPs) and the specific tissue inhibitors of metalloproteinases (TIMPs) are involved in tissue turnover in normal and pathological processes including wound healing. Marimastat, a potent inhibitor of MMPs, was used to investigate the role of MMPs in an in vitro wound contraction model, the dermal equivalent, in which fibroblasts are grown in a collagen matrix. Marimastat inhibited fibroblast-mediated lattice contraction and this inhibition was reversible upon removal of the inhibitor, indicating that MMPs play an important role in fibroblast-mediated collagen lattice contraction, modelling what may happen when granulation tissue contracts in a healing wound.

Lack of cyclin E immunoreactivity in non-malignant breast and association with proliferation in breast cancer.

Cyclin E is a G1 cyclin that is essential for the transition from G1 to S phase in the cell cycle. Alterations to cyclin E expression or regulation could be important in tumorigenesis. Previous immunohistochemical and immunoblotting studies have investigated the expression of cyclin E in breast carcinomas. In this study, cyclin E has been investigated in a range of non-malignant and malignant breast using immunohistochemistry. Normal and benign tissue from pre- and post-menopausal women (39 cases), non-involved tissue from cancer-containing breasts (47 cases), ductal carcinoma in situ (22 cases) and invasive breast carcinomas (109 cases) have been examined. There was no reactivity in any of the non-malignant breast. Only one ductal carcinoma in situ contained more than 5% reactive cells. A total of 28% of invasive carcinomas had > 5% of reactive cells (range 0-88% positive cells, mean 12.59%, median 1.0%). A significant association was found with poorer differentiation (P < 0.001), high MIB1 index (P < 0.001), lack of oestrogen receptor (0.05 > P > 0.025) and the presence of p53 protein (0.05 > P > 0.025). Virtually all cases with cyclin E and p53 were poorly differentiated. The presence of cyclin E is therefore only found in breast malignancies and is associated with more aggressive features, including high proliferation.

Hepatic adenosine triphosphate-dependent Ca2+ transport is mediated by distinct carriers on rat basolateral and canalicular membranes.

To characterize and localize hepatic plasma membrane ATP-dependent Ca2+ transport and Na+/Ca2+ exchange, studies were performed using highly purified rat basolateral and canalicular membrane vesicles. ATP-dependent Ca2+ transport activity was present in vesicles from both domains, insensitive to azide, oligomycin, oxalate, calmodulin, and calmidazolium, and virtually abolished at pH 6.8. However, basolateral and canalicular transport differed significantly. While basolateral transport was markedly stimulated by 1 mM Mg2+, canalicular transport was Mg2+ independent. Basolateral transport was similar at pH 7.4 and 8.0 but canalicular activity was stimulated fourfold at pH 8.0. Both Ca2+ Km [1.4 +/- 0.1 (SE).10(-8) vs. 4.8 +/- 0.7.10(-8) M] and Vmax (3.6 +/- 0.1 vs. 9.0 +/- 0.6 nmol mg-1 protein min-1) were lower in basolateral than in canalicular vesicles. Basolateral transport was somewhat more nucleotide specific (for ATP) and sensitive to vanadate (IC50 130 vs. 500 microM, respectively) than was canalicular transport. Na+/Ca2+ exchange activity was not detected in membranes from either domain. These studies suggest that hepatic ATP-dependent Ca2+ transport is mediated by domain-specific carriers on the basolateral and canalicular membranes.

Estrogenic potencies of resorcylic acid lactones and 17 beta-estradiol in female rats.

Uterotrophic response in sexually immature female rats has been used to rank the relative estrogenic potencies of six resorcylic acid lactones (RALs) and to compare their activities with that of 17 beta-estradiol. On oral administration, the estrogenic potency relative to 17 beta-estradiol is as follows: 7 alpha-zearalenol, 10 times less; zeranol, 150 times less; taleranol, 350 times less; zearalanone, 400 times less; zearalenone, 650 times less; 7 beta-zearalenol, 3500 times less. On subcutaneous administration, zeranol is 500 times less estrogenic than 17 beta-estradiol.

Hydroxyl/bile acid exchange. A new mechanism for the uphill transport of cholate by basolateral liver plasma membrane vesicles.

In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings suggest a specific carrier on the basolateral membrane of the hepatocyte which mediates hydroxyl/cholate exchange (or H+-cholate co-transport). A model for uphill cholate transport is discussed in which the Na+ pump would ultimately drive Na+/H+ exchange which in turn would drive hydroxyl/cholate exchange.

The isolation of nuclear envelopes. Effects of thiol-group oxidation and of calcium ions.

The effects of (a) oxidative cross-linking of protein thiol groups and (b) the presence or absence of Ca2+ ions on rat liver nuclear-envelope isolation were studied. Two envelope-isolation procedures were compared: a well characterized low-ionic-strength method and a recently developed high-ionic-strength method. The latter method seems preferable to the former in respect of lower intranuclear contamination of the envelopes, suppression of endogenous serine proteinase, and maintenance of high specific activities of envelope-associated enzymes. In both procedures, however, the presence of Ca2+ gave rise to a rapid, apparently irreversible, contamination of the envelopes by intranuclear material. This effect was half-maximal at 20 microM-Ca2+. In addition, the envelopes became contaminated with intranuclear material by a Ca2+-independent mechanism, apparently resulting from N-ethylmaleimide-sensitive intermolecular disulphide-bond formation. This oxidative process seemed to have two major kinetic components (half-life, t1/2, approx. 2 min and 10 min). In view of these findings, it is recommended that (i) for most purposes, nuclear envelopes be isolated by the newly developed high-ionic-strength procedure, (ii) irrespective of the method used, Ca2+-chelators be included in all the buffers, (iii) thiol-group oxidation be prevented or reversed during the procedure.