In silico Analysis Suggests Common Appearance of scaRNAs in Type II Systems and Their Association With Bacterial Virulence.

In addition to its well-established defense function, CRISPR/Cas can also exhibit crucial non-canonical activity through endogenous gene expression regulation, which was found to mainly affect bacterial virulence. These non-canonical functions depend on scaRNA, which is a small RNA encoded outside of CRISPR array, that is typically flanked by a transcription start site (TSS) and a terminator, and is in part complementary to another small CRISPR/Cas-associated RNA (tracrRNAs). Identification of scaRNAs is however largely complicated by the scarcity of RNA-Seq data across different bacteria, so that they were identified only in a relatively rare CRISPR/Cas subtype (IIB), and the possibility of finding them in other Type II systems is currently unclear. This study presents the first effort toward systematic detection of small CRISPR/Cas-associated regulatory RNAs, where obtained predictions can guide future experiments. The core of our approach is ab initio detection of small RNAs from bacterial genome, which is based on jointly predicting transcription signals - TSS and terminators - and homology to CRISPR array repeat. Particularly, we employ our improved approach for detecting bacterial TSS, since accurate TSS detection is the main limiting factor for accurate small RNA prediction. We also explore how our predictions match to available RNA-Seq data and analyze their conservation across related bacterial species. In Type IIB systems, our predictions are consistent with experimental data, and we systematically identify scaRNAs throughout this subtype. Furthermore, we identify scaRNA:tracrRNA pairs in a number of IIA/IIC systems, where the appearance of scaRNAs co-occurs with the strains being pathogenic. RNA-Seq and conservation analysis show that our method is well suited for predicting CRISPR/Cas-associated small RNAs. We also find possible existence of a modified mechanism of CRISPR-associated small RNA action, which, interestingly, closely resembles the setup employed in biotechnological applications. Overall, our findings indicate that scaRNA:tracrRNA pairs are present in all subtypes of Type II systems, and point to an underlying connection with bacterial virulence. In addition to formulating these hypotheses, careful manual curation that we performed, makes an important first step toward fully automated predictor of CRISPR/Cas-associated small RNAs, which will allow their large scale analysis across diverse bacterial genomes.

Attaining in vivo selectivity of positive modulation of α3ßγ2 GABAA receptors in rats: A hard task!

It is unclear whether GABAA receptors (GABAARs) that contain the α3-subunit are substantially involved in the anxiolytic effects of benzodiazepines (BDZs). In the present study, we tested YT-III-31, a newer BDZ ligand with functional preference for α3ßγ2 GABAARs, in two paradigms of unconditioned anxiety, the open field and elevated plus maze in rats. The effective dose of YT-III-31 (2 mg/kg) displayed a clear anxiolytic-like profile, unhampered by sedative action, in both tests. At a higher dose (10 mg/kg), YT-III-31 induced ataxia in the rotarod and sedation in spontaneous locomotor activity test. The latter effect was preventable by flumazenil and ßCCt, the non-selective and α1ßγ2 GABAAR affinity-selective antagonist, respectively, demonstrating that sedative properties of YT-III-31, when attained, are mediated by the α1γ2 site. To elucidate the receptor substrate of subtle behavioral differences between YT-III-31 and diazepam, we approximated in vivo receptor potentiation for both ligands, based on estimated unbound concentrations in rat brains. Far different from diazepam, YT-III-31 has significantly lower affinity for the α1γ2 over other BDZ-sensitive sites, and at lower doses (1-2 mg/kg) was devoid of potentiation at α1ßγ2 GABAARs. The approximation approach revealed a modest selectivity of YT-III-31 for α3γ2- in comparison to α2γ2 and α5γ2 binding sites, suggesting that its anxiolytic-like activity may not necessarily or predominantly reflect potentiation at α3ßγ2 GABAARs. Nonetheless, as the anxiolytic effects are achievable at a dose devoid of any sedative potential, and having favorable safety (cytotoxicity) and metabolic stability profile, YT-III-31 represents a valuable candidate for further translational research.

Quality of life in patients with MuSK positive myasthenia gravis.

It is believed that myasthenia gravis (MG) with antibodies to muscle-specific tyrosine kinase (MuSK) is the most severe form of the disease, especially in the first years of the disease. The aim of our study was to investigate quality of life (QoL) in a population of patients with MuSK MG compared to those with MG who have antibodies to acetylcholine receptor (AChR) in their sera. The study group consisted of 35 MuSK MG patients (28 females and 7 males), while the control group included 38 AChR MG patients matched for gender, age, and duration of the disease. SF-36 questionnaire was used to evaluate the health-related QoL. Following scales were also used: Hamilton's scales for depression and anxiety, the Multidimensional Scale of Perceived Social Support, and the Acceptance of Illness Scale. Physical domain scores of QoL were similarly affected in both MuSK and AChR groups, while mental domain and total SF-36 scores were even better in MuSK MG patients. Social support was better in the MuSK group (77.3 ± 9.3 vs. 70.6 ± 14.1, p < 0.05). SF-36 total score correlated with depression (rho = 0.54, p < 0.01), anxiety (rho = 0.49, p < 0.01), and MSPSS (rho = - 0.35, p < 0.05), and depression was an independent predictor of worse QoL. Besides therapy of weakness, psychiatric treatment and different forms of psychosocial condition should be part of regular therapeutic protocols for MG. Adequate team work of health professionals and family can provide a healthy mental environment in which a MuSK MG patient would feel more comfortable in spite of the disease.

Contribution of bacterial promoter elements to transcription start site detection accuracy.

Accurately detecting transcription start sites (TSS) is a starting point for understanding gene transcription, and an important ingredient in a number of applications necessary for functional gene annotation, such as gene and operon predictions. Available methods for TSS detection in bacteria use very different description of the bacterial promoter structure and all of them show low accuracy. It is therefore unclear which promoter features should be included in TSS recognition, and how their accuracy impacts the search detection. We here address this question for [Formula: see text] and [Formula: see text] (an alternative [Formula: see text] factor) promoters in E. coli. We find that [Formula: see text]35 element, which is considered exchangeable, and is often not included in TSS search, contributes to the search accuracy equally (for [Formula: see text], or more (for [Formula: see text] than the ubiquitous [Formula: see text]10 element. Surprisingly, the sequence of the spacer between [Formula: see text]35 and [Formula: see text]10 promoter elements, which is commonly included in TSS detection, significantly decreases the search accuracy for [Formula: see text] promoters. However, the spacer sequence improves the search accuracy for [Formula: see text] promoters, which we attribute to a presence of sequence conservation. Overall, there is as much as [Formula: see text]50% false positive reduction for optimally implemented promoter features in [Formula: see text], underlying necessity for accurate promoter element alignments.

Lipopolysaccharide exposure during late embryogenesis results in diminished locomotor activity and amphetamine response in females and spatial cognition impairment in males in adult, but not adolescent rat offspring.

Numerous basic and epidemiological studies have connected prenatal maternal immune activation with the occurrence of schizophrenia and/or autism. Depending on subtle differences in protocols of the used animal model, a variety of behavioral abnormalities has been reported. This study investigated behavioral differences in Wistar rat offspring of both genders, exposed to the 100 µg/kg per day dose of lipopolysaccharide (LPS) in late embryogenesis (embryonic days 15 and 16), while tested at their adolescent and young adult age (postnatal days 40 and 60, respectively). Immune activation was confirmed by detecting high levels of TNF-α and IL-6 in dam blood withdrawn 2h after the first dose of LPS. The animals were assessed in three consecutive trials of locomotor activity (novelty exploration, response to i.p. saline injection and challenge with 0.5mg/kg amphetamine), Morris water maze and social interaction tests. Overt behavioral dysfunction was perceived in adult rats only, and these changes were gender-distinctive. When compared with control rats, LPS females displayed baseline hypolocomotion and a decreased reactivity to amphetamine, while LPS males exhibited spatial learning (acquisition trials) and memory (probe trial) impairments. Prenatal treatment did not affect the time spent in social interaction. As maternal exposure to LPS in late gestation resulted in behavioral changes in offspring in early adulthood, it may model schizophrenia-like, but not autism-like endophenotypes. However, lack of a potentiated response to amphetamine testified that this model could not mimic positive symptoms, but rather certain traits of cognitive dysfunction and deficit symptoms, in males and females, respectively.

Negative modulation of α5 GABAA receptors in rats may partially prevent memory impairment induced by MK-801, but not amphetamine- or MK-801-elicited hyperlocomotion.

Reportedly, negative modulation of α5 GABAA receptors may improve cognition in normal and pharmacologically-impaired animals, and such modulation has been proposed as an avenue for treatment of cognitive symptoms in schizophrenia. This study assessed the actions of PWZ-029, administered at doses (2, 5, and 10 mg/kg) at which it reached micromolar concentrations in brain tissue with estimated free concentrations adequate for selective modulation of α5 GABAA receptors, in three cognitive tasks in male Wistar rats acutely treated with the noncompetitive N-methyl-d-aspartate receptor antagonist, MK-801 (0.1 mg/kg), as well in tests of locomotor activity potentiated by MK-801 (0.2 mg/kg) or amphetamine (0.5 mg/kg). In a hormetic-like manner, only 5 mg/kg PWZ-029 reversed MK-801-induced deficits in novel object recognition test (visual recognition memory), whereas in the Morris water maze, the 2 mg/kg dose of PWZ-029 exerted partial beneficial effects on spatial learning impairment. PWZ-029 did not affect recognition memory deficits in social novelty discrimination procedure. Motor hyperactivity induced with MK-801 or amphetamine was not preventable by PWZ-029. Our results show that certain MK-801-induced memory deficits can be ameliorated by negative modulation of α5 GABAA receptors, and point to the need for further elucidation of their translational relevance to cognitive deterioration in schizophrenia.