Prognostic value of the micronucleus assay for clinical endpoints in neoadjuvant radiochemotherapy for rectal cancer.

BACKGROUND: The question whether lymphocyte radiosensitivity is representative of patients' response to radiotherapy (RT) remains unsolved. We analyzed lymphocyte cytogenetic damage in patients who were homogeneously treated with preoperative radiochemotherapy (RCT) for rectal cancer within clinical trials. We tested for interindividual variation and consistent radiosensitivity after in-vivo and in-vitro irradiation, analyzed the effect of patients' and RCT characteristics on cytogenetic damage, and tested for correlations with patients' outcome in terms of tumor response, survival and treatment-related toxicity. METHODS: The cytokinesis-block micronucleus cytome (CBMNcyt) assay was performed on the peripheral blood lymphocytes (PBLCs) of 134 patients obtained before, during, at the end of RCT, and during the 2-year follow-up. A subset of PBLCs obtained before RCT was irradiated in-vitro with 3 Gy. RCT included 50.4 Gy of pelvic RT with 5-fluorouracil (5-FU) alone (n = 78) or 5-FU plus oxaliplatin (n = 56). The analyzed variables included patients' age, gender, RT characteristics (planning target volume size [PTV size], RT technique), and chemotherapy characteristics (5-FU plasma levels, addition of oxaliplatin). Outcome was analyzed as tumor regression, patient survival, and acute and late toxicity. RESULTS: Cytogenetic damage increased significantly with the radiation dose and varied substantially between individuals. Women were more sensitive than men; no significant age-dependent differences were observed. There was a significant correlation between the cytogenetic damage after in-vitro irradiation and in-vivo RCT. We found a significant effect of the PTV size on the yields of cytogenetic damage after RCT, while the RT technique had no effect. Neither the addition of oxaliplatin nor the 5-FU levels influenced cytogenetic damage. We found no correlation between patient outcome and the cytogenetic damage. CONCLUSIONS: We found consistent cytogenetic damage in lymphocytes after in-vivo RCT and in-vitro irradiation. Gender was confirmed as a well-known, and the PTV size was identified as a less well-known influencing variable on lymphocyte cytogenetic damage after partial-body irradiation. A consistent level of cytogenetic damage after in-vivo and in-vitro irradiation may indicate the importance of genetic factors for individual radiosensitivity. However, we found no evidence that in-vivo or in-vitro irradiation-induced cytogenetic damage is an adequate biomarker for the response to RCT in rectal cancer patients.

"Let's see what happens:"-Women's experiences of open-label placebo treatment for menopausal hot flushes in a randomized controlled trial.

Open-label (honestly prescribed) placebos are an ethical way to evoke placebo effects in patients. As part of a mixed-methods study, we conducted in-depth interviews with eight menopausal women who underwent and benefitted from open-label placebo treatment in a randomized-controlled trial of hot flushes. Data were analyzed using Interpretative Phenomenological Analysis. We found that the women had low expectations about the placebo treatment yet endorsed what they referred to as "hope" and openness to "see what happens". Recording hot flushes via the symptom diary was viewed as a valuable opportunity for self-examination and appraising outcomes. Receiving relief from the placebo treatment empowered women and enhanced their sense of control and agency. In summary, participants' initial openness towards placebos, their hopes to get better, monitoring symptoms closely, and taking the initiative to address symptoms were components of a positive open-label placebo experience.

Can physical exercise have a protective effect in an animal model of sleep-related movement disorder?

The purpose of the present study was to determine whether physical exercise (PE) has a protective effect in an experimental animal model of sleep-related movement disorder (A11 dopaminergic nuclei lesions with 6-OHDA). Rats were divided into four groups (Control PE-CTRL/PE, SHAM/PE, A11 lesion/NPE, A11 lesion/PE). Two experiments were performed: (1) the rats underwent PE before (2 weeks) and after (4 weeks) the A11 lesion; and (2) the rats underwent PE only after (4 weeks) the A11 lesion. Electrode insertion surgery was performed and sleep analyses were conducted over a period of 24h (baseline and after PE) and analyzed in 6 blocks of 4h. The results demonstrated that the A11 lesion produced an increased percentage of wakefulness in the final block of the dark period (3-7am) and a significant enhancement of the number of limb movements (LM) throughout the day. Four weeks of PE was important for reducing the number of LMs in the A11 lesion group in the rats that performed PE before and after the A11 lesion. However, in the analysis of the protective effect of PE on LM, the results showed that the number of LMs was lower at baseline in the group that had performed 2 weeks of PE prior to the A11 lesion than in the group that had not previously performed PE. In conclusion, these findings consistently demonstrate that non-pharmacological manipulations had a beneficial effect on the symptoms of sleep-related movement disorder.

Sleep-related movement disorder symptoms in SHR are attenuated by physical exercise and an angiotensin-converting enzyme inhibitor.

The relationship between hypertension and sleep-related movement disorders has been hypothesized for humans, but the causes and mechanisms have not been elucidated. We investigated whether an alteration in blood pressure (BP) induced by physical exercise and/or an angiotensin-converting enzyme inhibitor (enalapril) could affect locomotor activity in spontaneously hypertensive rats, with emphasis on the dopaminergic system. We used SHR and normotensive Wistar rats distributed into 4 groups for each strain: control, physical exercise, enalapril and physical exercise+enalapril. Physical exercise was performed on a treadmill, and enalapril was administered by gavage, both for 8weeks. During this period, locomotor activity was evaluated in an open field test, and BP was evaluated by tail plethysmography. Dopaminergic receptors, dopamine transporter and tyrosine hydroxylase levels at the striatum were evaluated by Western blotting. The control group of spontaneously hypertensive rats showed higher BP, increased activity in the open field test and lower levels of D2 receptors and tyrosine hydroxylase compared with all other groups throughout the experimental period. In general, physical exercise and enalapril attenuated these alterations. This study suggested the existence of comorbidity between hypertension and sleep-related movement disorders in spontaneously hypertensive rats. Physical exercise and enalapril conferred protection for both hypertension and the observed behavioral changes. In addition, these treatments led to changes in dopaminergic signaling in the striatal region (i.e., D2 receptor, TH and DAT).

Sleep pattern and locomotor activity are impaired by doxorubicin in non-tumor-bearing rats.

PURPOSE: We sought explore the effects of doxorubicin on sleep patterns and locomotor activity. To investigate these effects, two groups were formed: a control group and a Doxorubicin (DOXO) group. METHODS: Sixteen rats were randomly assigned to either the control or DOXO groups. The sleep patterns were examined by polysomnographic recording and locomotor activity was evaluated in an open-field test. RESULTS: In the light period, the total sleep time and slow wave sleep were decreased, while the wake after sleep onset and arousal were increased in the DOXO group compared with the control group (p<0.05). In the dark period, the total sleep time, arousal, and slow wave sleep were increased, while the wake after sleep onset was decreased in the DOXO group compared with the control group (p<0.05). Moreover, DOXO induced a decrease of crossing and rearing numbers when compared control group (p<0.05). CONCLUSIONS: Therefore, our results suggest that doxorubicin induces sleep pattern impairments and reduction of locomotor activity.

Presynaptic architecture of the larval zebrafish neuromuscular junction.

This article shows the ultrastructural architecture of larval zebrafish (Danio rerio) neuromuscular junctions in three dimensions. We compare classical electron microscopy fixation techniques with high-pressure freezing followed by freeze substitution (HPF/FS) in combination with electron tomography. Furthermore, we compare the structure of neuromuscular junctions in 4- and 8-dpf zebrafish larvae with HPF/FS because this allows for close-to-native ultrastructural preservation. We discovered that synaptic vesicles of 4-dpf zebrafish larvae are larger than those of 8-dpf larvae. Furthermore, we describe two types of dense-core vesicles and quantify a filamentous network of small filaments interconnecting synaptic vesicles as well as tethers connecting synaptic vesicles to the presynaptic cell membrane. In the center of active zones, we found elaborate electron-dense projections physically connecting vesicles of the synaptic vesicle pool to the presynaptic membrane. Overall this study establishes the basis for systematic comparisons of synaptic architecture at high resolution in three dimensions of an intact vertebrate in a close-to-native state. Furthermore, we provide quantitative information that builds the basis for diverse systems biology approaches in neuroscience, from comparative anatomy to cellular simulations.

Sleep disorders: A review of the interface between restless legs syndrome and iron metabolism.

Restless legs syndrome (RLS) is characterized by unpleasant sensations mainly in the legs. 43% of RLS-associated conditions have also been associated with systemic iron deficiency. The objective of this study was to review in the literature the relationship between iron metabolism and RLS. With an initial search using the keywords combination "Iron Metabolism OR Iron Deficiency AND Restless Legs Syndrome," 145 articles were screened, and 20 articles were selected. Few studies were found for this review in the period of 2001-2014, however, the correlation between RLS and iron was evident.

Changes in motor behavior during pregnancy in rats: the basis for a possible animal model of restless legs syndrome.

PURPOSE: Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS - sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features. METHODS: We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation. RESULTS: There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05). CONCLUSION: These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.

Changes in motor behavior during pregnancy in rats: the basis for a possible animal model of restless legs syndrome.

PURPOSE: Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS - sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features. METHODS: We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation. RESULTS: There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05). CONCLUSION: These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.

The effects of long-term dopaminergic treatment on locomotor behavior in rats.

Long-term treatments with dopaminergic agents are associated with adverse effects, including augmentation. Augmentation consists of an exacerbation of restless legs syndrome (a sleep-related movement disorder) symptoms during treatment compared to those experienced during the period before therapy was initiated. The objective of this study was to examine locomotor activity in rats after long-term dopaminergic treatment and its relationship with expression of the D2 receptor, in addition to demonstrating possible evidence of augmentation. The rats were divided into control (CTRL) and drug (Pramipexole-PPX) groups that received daily saline vehicle and PPX treatments, respectively, for 71 days. The locomotor behavior of the animals was evaluated weekly in the Open Field test for 71 days. The expression of the dopamine D2 receptor was evaluated by Western Blot analysis. The animals that received the PPX demonstrated a significant reduction in locomotor activity from day 1 to day 57 and a significant increase in immobility time from day 1 to day 64 relative to baseline values, but these values had returned to baseline levels at 71 days. No changes in the expression of the D2 receptor were demonstrated after treatment with a dopaminergic agonist. This study suggests changes in locomotor activity in rats after long-term PPX treatment that include an immediate reduction of locomotion and an increase in immobilization, and after 64 days, these values returned to baseline levels without evidence of augmentation. In addition, it was not possible to demonstrate a relationship between locomotor activity and the expression of D2 receptors under these conditions.

Spontaneously hypertensive rats: possible animal model of sleep-related movement disorders.

Clinical experience suggests that restless legs syndrome (RLS), periodic leg movement (PLM), and attention-deficit hyperactivity disorder (ADHD) may co-occur in both children and adults. The purpose of the present study was to provide an electrocorticography and electromyography evaluation of the spontaneously hypertensive rat (SHR) to investigate the potential of this rat strain as an animal model of RLS-PLM. Initial work focused on evaluating sleep patterns and limb movements during sleep in SHR, having normotensive Wistar rats (NWR) as control, followed by comparison of two treatments (pharmacological-dopaminergic agonist treatment and nonpharmacological-chronic physical exercise), known to be clinically beneficial for sleep-related movement disorders. The captured data strengthen the association between SHR and RLS-PLM, revealing a significant reduction on sleep efficiency and slow wave sleep and an increase on wakefulness and limb movements for the SHR group during the dark period, as compared to the NWR group, effects that have characteristics that are strikingly consistent with RLS-PLM. The pharmacological and nonpharmacological manipulations validated these results. The present findings suggest that the SHR may be a useful putative animal model to study sleep-related movement disorders mechanisms.

The effects of physical exercise on the serum iron profile in spontaneously hypertensive rats.

The purpose of this study was to evaluate the profile of serum iron in spontaneously hypertensive rats after an aerobic physical exercise. To accomplish this, 12 normotensive Wistar rats and 12 spontaneously hypertensive rats were distributed into "physical exercise" and "no physical exercise" groups. The animals in the physical exercise group underwent to an aerobic exercise for a total of 4 weeks. Blood was collected for the analysis of iron. Our results indicate that rats of the physical exercise group had significantly lower serum iron levels after the aerobic exercise protocol compared to the spontaneously hypertensive rats no physical exercise group (F ((3,16)) = 4.4915, p < 0.01). No significant difference was found between no physical exercise groups. The results indicated that the difference in iron may be due to an increased demand for iron, prompted by chronic physical exercise. In addition, erythrocytosis has been associated with increased blood pressure in spontaneously hypertensive rats, suggesting that iron reduction may be related to decreased blood pressure in these animals.

Changes in motor behavior during pregnancy in rats: the basis for a possible animal model of restless legs syndrome / Alterações no comportamento motor de ratas prenhes: bases para um possível modelo animal da síndrome das pernas inquietas

PURPOSE: Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS – sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features. METHODS: We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation. RESULTS: There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05). CONCLUSION: These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder. .

OBJETIVO: Mulheres grávidas apresentam de duas a trêz vezes maior probabilidade de desenvolver a Síndrome das Pernas Inquietas (SPI – distúrbio do movimento relacionado ao sono) do que a população geral. O objetivo do estudo foi avaliar o comportamento e a locomoção de ratas durante a gravidez, a fim de verificar se esses animais apresentam algumas características SPI-like. MÉTODOS: Foram utilizadas 14 fêmeas Wistar de 80 dias de idade, pesando entre 200 e 250 g. Os ratos foram distribuídos em grupos controle (CTRL) e prenhes (PN). Após uma avaliação inicial do comportamento e da atividade locomotora em um ambiente de campo aberto, o grupo PN foi introduzido a prenhez, e sua atividade e comportamento locomotor foram avaliados nos dias 3, 10 e 19 de prenhez e no período pós-lactação em paralelo ao grupo CTRL. Os níveis de ferro e transferrina séricos nos grupos CTRL e PN foram analisados em sangue coletado após eutanásia por decapitação. RESULTADOS: Não houve diferenças significativas na deambulação total, nos eventos de grooming, bolos fecais e urina entre os grupos CTRL e PN. No entanto, o grupo PN apresentou menos eventos de rearing, aumento no tempo de duração de grooming e redução no tempo de imobilização em relação ao grupo CTRL (ANOVA, p<0,05). CONCLUSÃO: Esses resultados sugerem que ratas prenhes apresentam alterações comportamentais e locomotoras semelhantes às observadas em modelos animais de SPI, demonstrando ser um possível modelo animal desse distúrbio do sono. .

Identification of the preprotein binding domain of SecA.

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase.

The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability. SecA Motif III mutants can bind ligands (nucleotide, the SecYEG translocase 'channel', signal and mature preprotein domains), can catalyse basal and SecYEG-stimulated ATP hydrolysis and can be activated for catalysis. However, Motif III mutation specifically blocks the preprotein-stimulated 'translocation ATPase' at a step of the reaction pathway that lies downstream of ligand binding. A functional Motif III is required for optimal ligand-driven conformational changes and kinetic parameters that underlie optimal preprotein-modulated nucleotide cycling at the SecA DEAD motor. We propose that helicase Motif III couples preprotein binding to the SecA translocation ATPase and that catalytic activation of SF2 enzymes through Motif-III-mediated action is essential for both polypeptide and nucleic-acid substrates.