Wildtype and A30P Mutant Alpha-Synuclein Form Different Fibril Structures

Parkinson’s Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT) fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied.     

Different cardiorespiratory responses to hemorrhage and hyperoxia in normotensive (WKY) and spontaneously hypertensive (SHR) rats

OBJECTIVE: To compare the cardiorespiratory responses underlying the beneficial effects of hyperoxia during blood loss between normotensive (WKY) and hypertensive (SHR) rats. METHODS: Experiments were carried out in anesthetized animals with both carotid bifurcations either innervated or denervated. The effects of breathing 60% O2 in N2 were studied either in combination with non-hypotensive hemorrhage or during hemorrhagic hypotension. RESULTS: In normoxia arterial pressure fell more in SHR than in WKY for a given blood loss. During hyperoxia, nerve-intact rats showed initial suppression of ventilation, but bifurcation-denervated rats a powerful enhancement. In all groups, hyperoxia increased the overall tone of venous capacitance vessels. CONCLUSIONS: The greater blood loss in SHR than in WKY when bleeding down to a given arterial pressure results from a stronger constriction of venous capacitance vessels. Hyperoxia improves the ability of the cardiorespiratory system to resist the effects of hemorrhage by increasing the overall venous tone, thus supporting cardiac filling, and in some cases also by increasing alveolar ventilation, probably secondary to improved cerebral oxygenation. The beneficial effects of hyperoxia were: (i) not prevented by carotid denervation, and thus were presumably direct tissue effects of oxygen, (ii) strikingly weaker in SHR than in normotensive (WKY) rats.     

Synthesis of novel N1-substituted bicyclic pyrazole amino acids and evaluation of their interaction with glutamate receptors

N1-substituted bicyclic pyrazole amino acids (S)-9a-9c and (R)-9a-9c, which are conformationally constrained analogues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested for activity at ionotropic and metabotropic glutamate receptors. Some of them turned out to be selective for the NMDA receptors, where they behaved as weak antagonists. The biological activity is mainly due to the interaction with the glutamate binding site, and not with the glycine co-agonist site.     

Micronutrients Involved in One-Carbon Metabolism and Risk of Breast Cancer Subtypes

Background

Vitamins involved in one-carbon metabolism are hypothesized to influence breast cancer (BC) risk. However, epidemiologic studies that examined associations between B vitamin intake and BC risk have provided inconsistent results. We prospectively examined, in the Italian ORDET cohort, whether B vitamin consumption was associated with risk of BC and BC subtypes.

Methods

After a mean follow-up of 16.5 years, 391 BCs were diagnosed among 10,786 cohort women. B vitamin intakes were estimated from food frequency questionnaires. Cox proportional hazard models adjusted for energy intake and confounders, estimated hazard ratios (HR) with 95% confidence intervals (CIs) for BC according to intake.

Results

RRs were 0.61 (95% CI 0.38–0.97 highest vs. lowest quartile; P trend 0.025) for thiamine; 0.48 (95% CI 0.32–0.71; P trend <0.001) for riboflavin; 0.59 (95% CI 0.39–0.90; P trend 0.008) for vitamin B6, and 0.65 (95% CI 0.44–0.95; P trend 0.021) for folate. As regards risk of BC subtypes, high riboflavin and folate were significantly associated with lower risk of estrogen receptor positive (ER+) and progesterone receptor positive (PR+) cancers, and high thiamine was associated with lower risk of ER-PR- cancers. High riboflavin was associated with lower risk of both HER2+ and HER2- cancers, high folate with lower risk of HER2- disease, and high thiamine with HER2+ disease.

Conclusions

These findings support protective effects of thiamine and one-carbon metabolism vitamins (folate, riboflavin, and vitamin B6) against BC in general; while folate may also protect against ER+PR+ and HER2- disease; and thiamine against ER-PR-, and HER2+ disease.     

Efficacy and acceptability of pharmacological,psychosocial, and brain stimulation interventions in children and adolescents with mental disorders: an umbrella review

Top‐tier evidence on the safety/tolerability of 80 medications in children/adolescents with mental disorders has recently been reviewed in this jour­nal. To guide clinical practice, such data must be combined with evidence on efficacy and acceptability. Besides medications, psychosocial inter­ventions and brain stimulation techniques are treatment options for children/adolescents with mental disorders. For this umbrella review, we systematically searched network meta‐analyses (NMAs) and meta‐analyses (MAs) of randomized controlled trials (RCTs) evaluating 48 medications, 20 psychosocial interventions, and four brain stimulation techniques in children/adolescents with 52 different mental disorders or groups of mental disorders, reporting on 20 different efficacy/acceptability outcomes. Co‐primary outcomes were disease‐specific symptom reduction and all‐cause discontinuation (“acceptability”). We included 14 NMAs and 90 MAs, reporting on 15 mental disorders or groups of mental disorders. Overall, 21 medications outperformed placebo regarding the co‐primary outcomes, and three psychosocial interventions did so (while seven outperformed waiting list/no treatment). Based on the meta‐analytic evidence, the most convincing efficacy profile emerged for amphetamines, methylphenidate and, to a smaller extent, behavioral therapy in attention‐deficit/hyperactivity disorder; aripiprazole, risperidone and several psychosocial interventions in autism; risperidone and behavioral interventions in disruptive behavior disorders; several antipsychotics in schizophrenia spectrum disorders; fluoxetine, the combination of fluoxetine and cognitive behavioral therapy (CBT), and interpersonal therapy in depression; aripiprazole in mania; fluoxetine and group CBT in anxiety disorders; fluoxetine/selective serotonin reuptake inhibitors, CBT, and behavioral therapy with exposure and response prevention in obsessive‐compulsive disorder; CBT in post‐traumatic stress disorder; imipramine and alarm behavioral intervention in enuresis; behavioral therapy in encopresis; and family therapy in anorexia nervosa. Results from this umbrella review of interventions for mental disorders in children/adolescents provide evidence‐based information for clinical decision making.     

Physical Interaction between MYCN Oncogene and Polycomb Repressive Complex 2 (PRC2) in Neuroblastoma: FUNCTIONAL AND THERAPEUTIC IMPLICATIONS*

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Strontium stratigraphy of the upper Miocene Lithothamnion Limestone in the Majella Mountain,central Italy,and its palaeoenvironmental implications

The ⁸⁷Sr/⁸⁶Sr isotope ratio has been widely used as a physical tool to date and correlate carbonate successions due to the long Sr residence time in comparison with the ocean mixing time. If this method works on oceanic successions, marginal basins may show different Sr isotope records in comparison with the coeval ocean one due to sea‐level variations, continental run‐off and restricted water exchanges. In this work, we present the ⁸⁷Sr/⁸⁶Sr isotope record of the upper Miocene carbonate ramp of the Lithothamnion Limestone (Majella Mountain, central Apennines), as an example of the onset of restricted water exchanges between a marginal basin and the ocean water masses. The overall latemost Tortonian–early Messinian Sr isotope record of the Lithothamnion Limestone fits below the global reference line. This deviation has been interpreted as due to the strong control that freshwater input and enhanced continental run‐off, linked to the migration of the Apennine accretionary wedge and foredeep system, have had on the central Adriatic water chemistry. These results imply that an accurate oceanographic and geodynamic framework along with diagenetic overprint investigation has to be taken into consideration prior to apply SIS on carbonate successions on marginal basins, even when facies analyses indicate fully marine conditions. This seems to be the case for the upper Miocene Central Mediterranean carbonate successions, but may have more general validity and be extended to other recent or past marginal basins.     

FSH: fast spaced seed hashing exploiting adjacent hashes

Background

Patterns with wildcards in specified positions, namely spaced seeds, are increasingly used instead of k-mers in many bioinformatics applications that require indexing, querying and rapid similarity search, as they can provide better sensitivity. Many of these applications require to compute the hashing of each position in the input sequences with respect to the given spaced seed, or to multiple spaced seeds. While the hashing of k-mers can be rapidly computed by exploiting the large overlap between consecutive k-mers, spaced seeds hashing is usually computed from scratch for each position in the input sequence, thus resulting in slower processing.

Results

The method proposed in this paper, fast spaced-seed hashing (FSH), exploits the similarity of the hash values of spaced seeds computed at adjacent positions in the input sequence. In our experiments we compute the hash for each positions of metagenomics reads from several datasets, with respect to different spaced seeds. We also propose a generalized version of the algorithm for the simultaneous computation of multiple spaced seeds hashing. In the experiments, our algorithm can compute the hashing values of spaced seeds with a speedup, with respect to the traditional approach, between 1.6\(\times\) to 5.3\(\times\), depending on the structure of the spaced seed.

Conclusions

Spaced seed hashing is a routine task for several bioinformatics application. FSH allows to perform this task efficiently and raise the question of whether other hashing can be exploited to further improve the speed up. This has the potential of major impact in the field, making spaced seed applications not only accurate, but also faster and more efficient.

Availability

The software FSH is freely available for academic use at: https://bitbucket.org/samu661/fsh/overview.