Dormant origins and fork protection mechanisms rescue sister forks arrested by transcription

The yeast RNA/DNA helicase Sen1, Senataxin in human, preserves the integrity of replication forks encountering transcription by removing RNA-DNA hybrids. Here we show that, in sen1 mutants, when a replication fork clashes head-on with transcription is arrested and, as a consequence, the progression of the sister fork moving in the opposite direction within the same replicon is also impaired. Therefore, sister forks remain coupled when one of the two forks is arrested by transcription, a fate different from that experienced by forks encountering Double Strand Breaks. We also show that dormant origins of replication are activated to ensure DNA synthesis in the proximity to the forks arrested by transcription. Dormant origin firing is not inhibited by the replication checkpoint, rather dormant origins are fired if they cannot be timely inactivated by passive replication. In sen1 mutants, the Mre11 and Mrc1-Ctf4 complexes protect the forks arrested by transcription from processing mediated by the Exo1 nuclease. Thus, a harmless head-on replication-transcription clash resolution requires the fine-tuning of origin firing and coordination among Sen1, Exo1, Mre11 and Mrc1-Ctf4 complexes

Sen1/SETX helicase limits DNA:RNA hybrids at DNA double strand breaks and determines faithful repair through end joining and homologous recombination

The repair of DNA double strand breaks (DSB) through non-homologous end joining (NHEJ) or homologous recombination (HR) is a finely regulated process. Genetic and molecular impedances in either of the pathways affect the fate of the DSB repair and can lead to extended chromosome rearrangements, triggering inheritable genetic instability and tumorigenesis. Recent findings indicated that formation of DNA:RNA hybrids at DSB sites might interfere with DSB repair and DNA damage response. Here we characterized the role of budding yeast DNA:RNA helicase Sen1, orthologous of the human Senataxin/SETX, in DSB repair. Chromatin immunoprecipitation analyses showed that Sen1 is recruited to one HO-induced DSB, contributing to remove DNA:RNA hybrids nearby the lesion. Along with the increased DNA:RNA hybrids, the binding of Rpb3, a subunit of the RNA polymerase II complex, is also prolonged in Sen1-depleted cells. Remarkably, the persistent DNA:RNA hybrids cause abrupt Mre11 and Dna2 dependent resection of the DSB, in the absence of Sen1. By specific genetic backgrounds, we found that Sen1 depletion leads to faster ectopic recombination repair of a DSB and, surprisingly, to elevated NHEJ and chromosome translocations. In line with the increased NHEJ, KU binding at the DSB is also prolonged in Sen1-depleted cells. In summary, our data suggest molecular mechanism through which Sen1 removes DNA:RNA hybrids at DSB in order to prevent non-canonical resection initiation, also limiting KU persistence at DSB and dangerous NHEJ events. We believed that these results in yeast might contribute to explain molecularly pathologic phenotypes, recently described in human cells depleted of Senataxin

Senataxin Ortholog Sen1 Limits DNA:RNA Hybrid Accumulation at DNA Double-Strand Breaks to Control End Resection and Repair Fidelity

Rawal et al. identify a non-canonical mechanism of DSB processing in yeast cells lacking the Senataxin ortholog Sen1 helicase, which strictly depends on DNA:RNA hybrid formation at the break and is associated with unfaithful DSB repair and genome instability. An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity

Blood pressure during nocturnal sleep in headache

AIM: Interactions between blood pressure control, sleep and headache have been largely studied, although not well understood. We designed a study trying to simultaneously evaluate all three aspects in the same subjects. We particularly concentrated on the observation of physiological blood pressure circadian rhythm, and the presence of cutaneous allodynia correlated to headache. Objective of the study was to investigate blood pressure during nocturnal sleep in patients that underwent a blood pressure 24 hours monitoring, and at the same time the presence of headache and of sleep behavioural alterations. METHODS: Blood pressure 24 hours monitoring was performed by an ambulatory blood pressure (ABP) monitor (Space Labs) with its ad hoc software. Headache diagnosis was made according to ICHD-II criteria. Presence of allodynia and sleep behavior were evaluated through semi-structured ad hoc questionnaires. RESULTS: A total of 195 subjects were included, of which 122 without headache (mean age 60.4\ub111.6 years, 78 men and 44 women) and 73 with history of headache, (mean age 54.2\ub112.5 years, 18 men and 55 women). Fifty-one headache patients had migraine (mean age 52.6\ub111.7 years, 11 men and 40 women) and 22 tension type headache (TTH - mean age 58.0\ub113.5 years, 7 men and 15 women). Allodynia was found in 30 out of 73 headache patients: 23 out of 51 in the migraine group and in 7 out of 22 in the tension-type one. The physiological reduction of blood pressure during night (dipping) was more conserved among headache patients (34 dippers out of 73 subjects, 46,6%) with respect to subjects without headache (40 dippers out of 122, 32,8%) and that this border-line difference was more strongly significant comparing allodynic subjects (19 dippers out of 30, 63.3%) with both non-headache (40 dippers out of 122, 32.8%, P<0.001) and non-allodynic (15 out of 43, 34.9%, P<0.05) ones. No significant difference was observed between headache patients and subjects without headache in terms of mean systolic and diastolic pressure, neither between migraine and TTH. CONCLUSION: Allodynic headache patients seem to maintain a more physiologic pressure circadian rhythm. While considering the possibility of selection bias, the hypothesis of an allostatic function of headache and allodynia in patients with unbalanced blood pressure could be made

Possible relationships between headache-allodynia and nocturnal sleep breathing

Sleep and headache are linked in a bidirectional way. Breathing quality during sleep may be a possible link between them. The objective of this study were to evaluate the prevalence of headache-and of allodynia-in a population of subjects who underwent cardiopulmonary monitoring during sleep for presumed respiratory problems; to evaluate the possible relationships between the presence of headaches-and of allodynia-and respiratory parameters. We studied 181 subjects, 112 without headache (mean age 59.4 +/- A 13.1 years, 97 men and 15 women); 69 with history of headache (42 men and 27 women; 41 migraineurs and 28 with tension type headache). Headache diagnosis was made according to ICHD-II criteria. A semi-structured ad hoc questionnaire was used to evaluate the presence of allodynia. Full cardiopulmonary monitoring was performed by SOMNO check(A (R)) effort (WEINMANN) with SaO(2), T90 and AHI determination. Headache and headache-associated allodynia were particularly frequent in this population, suggesting a positive correlation between breathing problems during sleep and head pain, and allodynia. The observation that better respiratory parameters were found among headache sufferers with respect to those without headache, even in allodynic subjects, seems to reverse this point of view: headache and allodynia may possibly have an allostatic function preventing deep sleep and, in turn, avoiding prolonged apneas

Possible correlations between blood pressure, primary headaches and cutaneous allodynia

Following an allostatic perspective, episodic migraine (M) may be considered as an adaptive behavioural response to endogenous or exogenous stressors, while its progression to a daily or nearly daily form (chronic migraine) may represent the failure of adaptive strategies. Multiple factors may enhance the progression/chronification of M, and among these the presence of cutaneous allodynia (CA) as well as alterations in blood pressure and in sleep. The working hypothesis of the study was that subjects with M, and particularly those with CA, could show a tendency towards high blood pressure levels and/or to alterations in the circadian rhythm of blood pressure. We studied 235 subjects consecutively attending a centre for blood pressure control for a blood pressure 24 h monitoring. Headache diagnosis was made according to the ICHD-II criteria. The presence of CA was evaluated through a semi-structured ad hoc questionnaire. Blood pressure 24 h monitoring was performed by an ambulatory blood pressure monitor (Space Labs) with its ad hoc software. Seventy-eight subjects had a history of headache (mean age 54.0 \ub1 12.4 years, 18 men and 60 women); 56 of them had M, 22 had tension-type headache; among them, CA was found in 24/56 subjects with M, and in 6/22 with tension-type headache; 157 subjects did not suffer from headache (mean age 60.5 \ub1 11.5 years, 99 men and 58 women). No significant difference was observed between headache subjects and subjects without headache in terms of mean systolic and diastolic pressure, neither in the M nor in tension-type subgroups. With regard to the circadian rhythm of blood pressure, the physiological reduction during night (dipping) was more evident among headache subjects than in subjects without headache; this border-line difference was more strongly significant in subjects with CA than both non-headache (p = 0.003) and non-CA (p = 0.05) ones. The difference between allodynic and non-allodynic subjects was present also in the M sub-group (7 dippers out of 32 non-allodynic migraineurs vs. 12 dippers out of 24 allodynic migraineurs, p = 0.03) notwithstanding the reduction of the sample size. Despite the initial hypothesis, subjects with primary headaches did not show differences in terms of mean blood pressure values and they showed a more physiologic blood pressure daily rhythm than those without headaches. Also the presence of CA, a marker of progression to chronic headache forms, was associated neither with hypertension nor with increased frequency of loss of dipping. M, particularly when associated with allodynia, may improve breathing during nocturnal sleep and consequently counteract possible blood pressure alterations, suggesting an allostatic function of allodynic headache

Anticonvulsant effects and behavioural outcomes of rAAV serotype 1 vector-mediated neuropeptide Y overexpression in rat hippocampus

Neuropeptide Y (NPY) is an endogenous peptide with powerful anticonvulsant properties. Its overexpression in the rat hippocampus, mediated by the local application of recombinant adeno-associated viral (rAAV) vectors carrying the human NPY gene, results in significant reduction of seizures in acute and chronic seizure models. In this study, we characterized a more efficient rAAV-NPY vector to improve cell transfection in the injected area. The changes included pseudotyping with the AAV vector serotype 1 (rAAV1), and using the strong constitutive hybrid CBA promoter, which contains a cytomegalovirus enhancer and chicken beta-actin promoter sequences. We compared NPY expression and the associated anticonvulsant effects of this new vector, with those mediated by the former rAAV vector with chimeric serotype 1/2 (rAAV1/2). In addition, we investigated whether rAAV serotype 1 vector-mediated chronic NPY overexpression causes behavioural deficits that may detract from the clinical utility of this therapeutic approach. We report that rAAV-NPY serotype 1 vector has significantly improved anticonvulsant activity when compared with serotype 1/2 vector, as assessed by measuring EEG seizure activity in kainic acid treated rats. rAAV1-mediated NPY overexpression in naive rats did not result in alterations of physiological functions such as learning and memory, anxiety and locomotor activity. In addition, we did not observe glia activation, or humoral immune responses against serotype 1 vector, which could inactivate gene expression. Our findings show that rAAV1-NPY vector with the CBA promoter mediates powerful anticonvulsant effects and seems to be safe in rodents, thus it may be considered a vector of choice for possible clinical applications

An atypical presentation of diffuse midline pontine glioma in a middle age patient: Case report

Introduction: Diffuse midline glioma is a newly WHO defined entity (grade IV) (Louis et al., 2016) which includes diffuse intrinsic pontine glioma (DIPG) reported in pediatric population and, occasionally, in young adults. Here, we present a detailed description of an atypical case of diffuse midline glioma in a 53 years old woman. Case report: A caucasian woman aged 53 from Ukraine, was referred to another neurological department complaining of 3 months history of progressive postural instability and gait impairment with frequent falling. Magnetic resonance demonstrated two brainstem lesions, hyperintense in FLAIR with \u201cpatchy\u201d peripheral enhancement, leptomeningeal and cranial nerves enhancement. CSF was normal. Due to positive antinuclear antibodies test (ANA 1:360), intravenous steroid treatment was administered and reported to initially improve the patient condition. However, the following weeks the lady worsened. Imaging features were unchanged. Because quantiferon test resulted positive, MRI-Spectroscopy showed an inflammatory pattern and MRI perfusion study and brain FDG-PET, were normal, tubercolar granulomatous hypothesis was initially favored. Antitubercular therapy with isoniazid, pyrazinamide, ethambutol and rifampicin was started without any clinical improvement. Hence, the biopsy was proposed. The procedure revealed a diffuse midline pontine glioma. Considering the advanced stage of the disease, radiotherapy was not indicated. Patient died after eight months from the onset of neurological disturbances. Conclusion: Our case shows that diffuse midline glioma is a CNS tumor not limited to young population but occurring also in middle aged patients with an insidious pattern. We therefore recommend to perform biopsy at very early stages in patients with atypical brainstem lesions