Long non‐coding RNAs in melanoma

Melanoma is the most lethal cutaneous cancer with a highly aggressive and metastatic phenotype. While recent genetic and epigenetic studies have shed new insights into the mechanism of melanoma development, the involvement of regulatory non‐coding RNAs remain unclear. Long non‐coding RNAs (lncRNAs) are a group of endogenous non‐protein‐coding RNAs with the capacity to regulate gene expression at multiple levels. Recent evidences have shown that lncRNAs can regulate many cellular processes, such as cell proliferation, differentiation, migration and invasion. In the melanoma, deregulation of a number of lncRNAs, such as HOTAIR, MALAT1, BANCR, ANRIL, SPRY‐IT1 and SAMMSON, have been reported. Our review summarizes the functional role of lncRNAs in melanoma and their potential clinical application for diagnosis, prognostication and treatment.     

Factors related to gizzard shad and the threadfin shad occurrence and abundance in Florida lakes

Gizzard shad Dorosoma cepedianum were collected in 23 and threadfin shad D. petenense were collected in 22 of the 60 Florida lakes sampled. Logistic regression equations were 94% effective for predicting gizzard shad occurrence from chlorophyll and lake surface area, and 84% effective for predicting threadfin shad occurrence from lake surface area and lake volume inhabited (PVI). Occurrence of both shad species was related positively to lake size. In lakes where gizzard shad or threadfin shad were collected, shad density and biomass of both shad species were related positively to chlorophyll. Gizzard shad populations were generally vulnerable to predation in lakes, with the per cent of gizzard shad ≤200mm L _T values exceeding 60% with few exceptions. Effects of gizzard shad and threadfin shad on fish community dynamics may be confined to relatively large (>100 ha) and fertile (chlorophyll >20–30μg l⁻¹) Florida lakes.     

Changes in odor quality discrimination following recovery from olfactory nerve transection

Following recovery from olfactory nerve transection, animals regain their ability to discriminate between odors. Odor discrimination is restored after new neurons establish connections with the olfactory bulb. However, it is not known if the new connections alter odor quality perception. To address this question, 20 adult hamsters were first trained to discriminate between cinnamon and strawberry odors. After reaching criterion (> or = 90% correct response), half of the animals received a bilateral nerve transection (BTX) and half a surgical sham procedure. Animals were not tested again until day 40, a point in recovery when connections are re-established with the bulb. When BTX animals were tested without food reinforcement, they could not perform the odor discrimination task. Sham animals, however, could discriminate, demonstrating that the behavioral response had not been extinguished during the 40 day period. When reinforcement was resumed, BTX animals were able to discriminate between cinnamon and strawberry after four test sessions. In addition, their ability to discriminate between these two familiar odors was no different than that of BTX and sham animals tested with two novel odors, baby powder and coffee. These findings suggest that, after recovery from nerve transection, there are alterations in sensory perception and that restoration of odor quality discrimination requires that the animal must again learn to associate individual odor sensations with a behavioral response.      

Glutamate metabolism in cerebral mitochondria after ischemia and post‐ischemic recovery during aging: relationships with brain energy metabolism

Glutamate is involved in cerebral ischemic injury, but its role has not been completely clarified and studies are required to understand how to minimize its detrimental effects, contemporarily boosting the positive ones. In fact, glutamate is not only a neurotransmitter, but primarily a key metabolite for brain bioenergetics. Thus, we investigated the relationships between glutamate and brain energy metabolism in an in vivo model of complete cerebral ischemia of 15 min and during post‐ischemic recovery after 1, 24, 48, 72, and 96 h in 1‐year‐old adult and 2‐year‐old aged rats. The maximum rates (V _max) of glutamate dehydrogenase (GlDH ), glutamate‐oxaloacetate transaminase, and glutamate‐pyruvate transaminase were assayed in somatic mitochondria (FM ) and in intra‐synaptic ‘Light’ mitochondria and intra‐synaptic ‘Heavy’ mitochondria ones purified from cerebral cortex, distinguishing post‐ and pre‐synaptic compartments. During ischemia, none of the enzymes were modified in adult animals. In aged ones, glutamate‐oxaloacetate transaminase was increased in FM and GlDH in intra‐synaptic ‘Heavy’ mitochondria, stimulating glutamate catabolism. During post‐ischemic recovery, FM did not show modifications at both ages while, in intra‐synaptic mitochondria of adult animals, glutamate catabolism was increased after 1 h of recirculation and decreased after 48 and 72 h, whereas it remained decreased up to 96 h in aged rats. These results, with those previously published about Krebs’ cycle and Electron Transport Chain (Villa et al ., [2013] Neurochem. Int . 63, 765–781), demonstrate that: (i) V _max of energy‐linked enzymes are different in the various cerebral mitochondria, which (ii) respond differently to ischemia and post‐ischemic recovery, also (iii) with respect to aging.

     

Octopamine in male aggression of Drosophila

BACKGROUND: In mammals and humans, noradrenaline is a key modulator of aggression. Octopamine, a closely related biogenic amine, has been proposed to have a similar function in arthropods. However, the effect of octopamine on aggressive behavior is little understood. RESULTS: An automated video analysis of aggression in male Drosophila has been developed, rendering aggression accessible to high-throughput studies. The software detects the lunge, a conspicuous behavioral act unique to aggression. In lunging, the aggressor rears up on his hind legs and snaps down on his opponent. By using the software to eliminate confounding effects, we now show that aggression is almost abolished in mutant males lacking octopamine. This suppression is independent of whether tyramine, the precursor of octopamine, is increased or also depleted. Restoring octopamine synthesis in the brain either throughout life or in adulthood leads to a partial rescue of aggression. Finally, neuronal silencing of octopaminergic and tyraminergic neurons almost completely abolishes lunges. CONCLUSIONS: Octopamine modulates Drosophila aggression. Genetically depleting the animal of octopamine downregulates lunge frequency without a sizable effect on the lunge motor program. This study provides access to the neuronal circuitry mediating this modulation.     

Effects of prostaglandin E2 (PGE2) on estradiol-17 beta-induced luteolysis in the nonpregnant ewe

Fifteen ewes were assigned as they came into estrus to the following randomized treatment groups: 1) Vehicle (1 ml corn oil + vehicle Na2CO3 buffer), 2) Estradiol-17 beta + vehicle and 3) Estradiol-17 beta + PGE2 (500 micrograms) in Na2CO3 buffer (5 ewes/treatment group). Prostaglandin E2 was given through an intrauterine cannula every four hours from days 8 through 15 postestrus. PGE2 prevented a luteolytic dose of estradiol-17 beta given on days 9 and 10 from causing a precocious luteolysis. PGE2 maintained concentrations of progesterone in peripheral blood (days 8 through 15) and weights and concentrations of progesterone in corpora lutea on day 15 postestrus of ewes receiving estradiol-17 beta. It is concluded that chronic intrauterine infusions of PGE2 can prevent an estradiol-17 beta-induced premature luteolysis.     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture

Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2-12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p greater than 0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p less than 0.05) with a 24-h pulse of dibutyryl adenosine 3', 5'-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal: Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3-12) resulted in continuously stimulated (p less than 0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p less than 0.05) percentage of cells staining positive for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase (3 beta HSD) activity were recovered on Day 12 in cultures incubated (Days 3-12) with dbcAMP. Incubation of cultures continuously with prostaglandin F2 alpha (PGF2 alpha) produced dose-dependent inhibition (p less than 0.05) of progesterone secretion. Reduced numbers of 3 beta HSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF2 alpha) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.     

Comparison of subpopulations of luteal cells obtained from cyclic and superovulated ewes

Peripheral blood samples were collected daily (Days 1-10 after ovulation) and analysed for progesterone content. Luteal tissue was collected on Day 10 after the LH surge, or Day 10 after hCG injection from cyclic and superovulated ewes, respectively. The tissue was enzymically dispersed and an aliquant was utilized for measurement of cell diameters, and staining for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase activity (3 beta-HSD). The remaining cell preparation was separated into small (10-22 micron) and large (greater than 22 micron) cell fractions by elutriation. Small and large cell suspensions were incubated (37 degrees C, 2 h) in the presence or absence or ovine LH (100 ng/ml) or dbcAMP (2 mM) and progesterone content of the medium was measured. Superovulation did not affect circulating progesterone concentrations, when expressed per mg luteal tissue recorded; basal progesterone production by small or large luteal cells; the unresponsiveness of large luteal cells to ovine LH or dbcAMP; the ratio of small:large cells recovered by dissociation the mean diameter of total cells; or the mean diameter of large cells. However, the mean cell diameter and LH stimulation of progesterone production by small cells were greater (P less than 0.05) in luteal tissue collected from superovulated than in that from cyclic ewes. These differences appear to be an amplification of basic function. Therefore, we conclude that corpora lutea obtained from superovulated ewes can be used to study functional aspects of small and large cells.