Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

The cAMP responsive element-binding (CREB)-1 gene increases risk of major psychiatric disorders.

Bipolar disorder (BPD), schizophrenia (SCZ) and unipolar major depressive disorder (MDD) are primary psychiatric disorders sharing substantial genetic risk factors. We previously reported that two single-nucleotide polymorphisms (SNPs) rs2709370 and rs6785 in the cAMP responsive element-binding (CREB)-1 gene (CREB1) were associated with the risk of BPD and abnormal hippocampal function in populations of European ancestry. In the present study, we further expanded our analyses of rs2709370 and rs6785 in multiple BPD, SCZ and MDD data sets, including the published Psychiatric Genomics Consortium (PGC) genome-wide association study, the samples used in our previous CREB1 study, and six additional cohorts (three new BPD samples, two new SCZ samples and one new MDD sample). Although the associations of both CREB1 SNPs with each illness were not replicated in the new cohorts (BPD analysis in 871 cases and 1089 controls (rs2709370, P=0.0611; rs6785, P=0.0544); SCZ analysis in 1273 cases and 1072 controls (rs2709370, P=0.230; rs6785, P=0.661); and MDD analysis in 129 cases and 100 controls (rs2709370, P=0.114; rs6785, P=0.188)), an overall meta-analysis of all included samples suggested that both SNPs were significantly associated with increased risk of BPD (11 105 cases and 51 331 controls; rs2709370, P=2.33 × 10-4; rs6785, P=6.33 × 10-5), SCZ (34 913 cases and 44 528 controls; rs2709370, P=3.96 × 10-5; rs6785, P=2.44 × 10-5) and MDD (9369 cases and 9619 controls; rs2709370, P=0.0144; rs6785, P=0.0314), with the same direction of allelic effects across diagnostic categories. We then examined the impact of diagnostic status on CREB1 mRNA expression using data obtained from independent brain tissue samples, and observed that the mRNA expression of CREB1 was significantly downregulated in psychiatric patients compared with healthy controls. The protein-protein interaction analyses showed that the protein encoded by CREB1 directly interacted with several risk genes of psychiatric disorders identified by GWAS. In conclusion, the current study suggests that CREB1 might be a common risk gene for major psychiatric disorders, and further investigations are necessary.

The protocadherin 17 gene affects cognition, personality, amygdala structure and function, synapse development and risk of major mood disorders.

Major mood disorders, which primarily include bipolar disorder and major depressive disorder, are the leading cause of disability worldwide and pose a major challenge in identifying robust risk genes. Here, we present data from independent large-scale clinical data sets (including 29 557 cases and 32 056 controls) revealing brain expressed protocadherin 17 (PCDH17) as a susceptibility gene for major mood disorders. Single-nucleotide polymorphisms (SNPs) spanning the PCDH17 region are significantly associated with major mood disorders; subjects carrying the risk allele showed impaired cognitive abilities, increased vulnerable personality features, decreased amygdala volume and altered amygdala function as compared with non-carriers. The risk allele predicted higher transcriptional levels of PCDH17 mRNA in postmortem brain samples, which is consistent with increased gene expression in patients with bipolar disorder compared with healthy subjects. Further, overexpression of PCDH17 in primary cortical neurons revealed significantly decreased spine density and abnormal dendritic morphology compared with control groups, which again is consistent with the clinical observations of reduced numbers of dendritic spines in the brains of patients with major mood disorders. Given that synaptic spines are dynamic structures which regulate neuronal plasticity and have crucial roles in myriad brain functions, this study reveals a potential underlying biological mechanism of a novel risk gene for major mood disorders involved in synaptic function and related intermediate phenotypes.

Role of the Streptococcus mutans CRISPR-Cas systems in immunity and cell physiology.

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

Genome-wide survey implicates the influence of copy number variants (CNVs) in the development of early-onset bipolar disorder.

We used genome-wide single nucleotide polymorphism (SNP) data to search for the presence of copy number variants (CNVs) in 882 patients with bipolar disorder (BD) and 872 population-based controls. A total of 291 (33%) patients had an early age-at-onset < or =21 years (AO < or =21 years). We systematically filtered for CNVs that cover at least 30 consecutive SNPs and which directly affect at least one RefSeq gene. We tested whether (a) the genome-wide burden of these filtered CNVs differed between patients and controls and whether (b) the frequency of specific CNVs differed between patients and controls. Genome-wide burden analyses revealed that the frequency and size of CNVs did not differ substantially between the total samples of BD patients and controls. However, separate analysis of patients with AO < or =21 years and AO>21 years showed that the frequency of microduplications was significantly higher (P=0.0004) and the average size of singleton microdeletions was significantly larger (P=0.0056) in patients with AO < or =21 years compared with controls. A search for specific BD-associated CNVs identified two common CNVs: (a) a 160 kb microduplication on 10q11 was overrepresented in AO < or = 21 years patients (9.62%) compared with controls (3.67%, P=0.0005) and (b) a 248 kb microduplication on 6q27 was overrepresented in the AO< or = 21 years subgroup (5.84%) compared with controls (2.52%, P=0.0039). These data suggest that CNVs have an influence on the development of early-onset, but not later-onset BD. Our study provides further support for previous hypotheses of an etiological difference between early-onset and later-onset BD.

The association between endometrial polyps, chronic endometritis, and IVF outcomes.

OBJECTIVE: Endometrial polyps (EPs) are one of the most common pathologies detected during the examination of the uterine cavity of infertile women. We aimed to demonstrate the relationship between EPs, chronic endometritis (CE) and in vitro fertilization (IVF) outcomes. PATIENTS AND METHODS: This retrospective study was performed on 394 hysteroscopically examined infertility cases. We performed polyp resections (PR) and extensive biopsies of the endometrium to demonstrate the association with clinical pregnancy (CP) by IVF. We performed statistical analysis to compare these associations. RESULTS: The incidence of CE was twice as high in the presence of EPs as in the absence of EPs. The associations between EPs and PR were found to be significant for positive CP outcomes. A significant difference in IVF outcome was found between the group with EPs and the group without EPs. All these associations were statistically significant (p < 0.05). CONCLUSIONS: We found a frequent association between EPs and CE. The pregnancy rate obtained after IVF was negatively affected by the presence of EPs. Treatment of these pathologies improved IVF outcomes.

The Role of Early Diagnosis of Hepatorenal Cystic (HRC) Syndrome in Children-Clinical Trial.

The hepatorenal cystic (HRC) syndrome is a heterogeneous group of severe monogenic conditions that may be detected before birth. Effective programme evaluation of children with HRC syndrome is a systematic way to identify the renal and urinary tract malformations which represent the most common cause of end-stage renal disease (ESRD). We conducted a study involving 50 patients, who were between 3 months and 16 years of age, with multiple admissions in the Nephrology Department of "Maria Sklodowska Curie" Children Emergency Hospital from Bucharest, during 6 years (April 14th 2010-October 24th 2016), to evaluate the HRC syndrome. The admission symptomatology was mainly represented by the nephrology evaluation which was essential in the management of children's polycystic kidney disease. For example, a premature infant (gestational age=32 weeks) with positive heredo-collateral history (mother and grandmother were diagnosed with polycystic kidney disease), was tested positive for cystic renal disease after the fetal morphology was performed. It was also done a genetic determination for the presence of PKD1 and PKD2 mutations which are specific to autosomal dominant polycystic kidney disease-ADPKD. However, the genetic test was negative and a postnatal nephrological evaluation was performed using renal ultrasound. The image revealed autosomal recessive polycystic kidney disease-ARPKD. This study emphasizes the importance of an early diagnosis (prenatal, neontal, postnatal) correlated with the admission symptoms and also with the genetic diagnosis (mutations of PKD1 and PKD2).

Patterns of parental transmission and familial aggregation models in bipolar affective disorder.

Two recent studies [McMahon et al., 1995: Am J Hum Genet 56:1277-1286; Gershon et al., 1996: Am J Med Genet (Neuropsychiatr Genet) 67:202-207] reported an excess of maternal transmission in bipolar affective disorder in multiply affected families. In a sample of 130 families ascertained through a bipolar proband without regard to psychiatric family history we analysed the frequency of maternal (MAT) and paternal (PAT) transmissions, the morbid risk (MR) in relatives of transmitting mothers and fathers and the inheritance patterns in families with MAT vs. PAT transmission of the disease. In the total sample of 130 families we identified 39 families where the disease was transmitted from the paternal side (PAT families) and 35 families where the disease was transmitted from the maternal side (MAT families). Counting PAT and MAT transmissions in these unilineal families we found nearly equal numbers for both transmission types under a narrow (BP: bipolar disorder, schizoaffective-bipolar type disorder) and a broad definition (AFF: BP, recurrent unipolar depression) of the phenotype. The MRs for narrow and broad phenotypes were not significantly different in any type of PAT relative in PAT families vs. MAT relatives in MAT families. However, in PAT families there were two times more affected females than males with both disease models, while in MAT families there was no MR difference by relatives' sex. The transmission of BP was compatible with the Mendelian major gene model in PAT families and with the multifactorial model in MAT families. Extension of the relatives' phenotype led to borderline non-Mendelian major effects in PAT families and reproduced the multifactorial model in MAT families.

Different familial transmission patterns in bipolar I disorder with onset before and after age 25.

Gene identification in common disorders such as Alzheimer disease and breast cancer has greatly profited from the use of age of onset as criterion to delineate subgroups of disease characterized by different inheritance patterns. In bipolar affective disorder, where the majority of linkage studies have produced conflicting results, studies reporting clinical characteristics and familial occurrence of disease have suggested that age of onset might serve as an indicator for identifying more homogeneous subgroups of disease. Our study was the first to examine this hypothesis by the means of segregation analysis. We investigated a sample of 177 bipolar I probands recruited from consecutive admissions and their first- and second-degree relatives (2,407 subjects). Probands were subdivided into an early-onset (n = 107) and a late-onset group (n = 70) using an age of onset of 25 as a cut-off point. This age was chosen because the observed age of onset distribution was bimodal with a cut-off of 25 years. Morbid risks for affective disorder were found significantly higher (P = 0.01) in relatives of probands with an early onset than in probands with late onset of disease. The segregation analysis showed that the disease is transmitted differently in early- and late-onset groups. In the early-onset group, a non-Mendelian major gene with a polygenic component was favored while the data in the late-onset group were compatible with a multifactorial model. This result may have important implications for future molecular studies aiming at the identification of disease-associated genes.

Psychopathology in children aged 10-17 of bipolar parents: psychopathology rate and correlates of the severity of the psychopathology.

Seventy-two proband children aged 10-17 of bipolar parents, matched with 72 control children of normal parents, were investigated using DSM-III diagnostic criteria and multiple sources of information. The psychopathology rate in children (61% in probands versus 25% in controls) was related to the impact of psychic disorders on the children's adaptive functioning. The effect of several variables describing the psychiatric status of both parents and familial environment on the severity of psychopathology in children was analysed. Disordered and non-disordered probands were compared with respect to illness characteristics of their parents, familial environment, personality traits, and IQ by means of canonical discriminant analysis.

Adolescent offspring of endogenous unipolar depressive parents and of normal parents.

Ninety-six children aged 10-17 of unipolar endogenous depressive proband parents and 96 matched control children of well parents were investigated using DSM-IIIR diagnostic criteria. Both sets of parents were also studied. Although the rate of psychopathology was significantly higher in proband than in control children, adaptive functioning as a measure of the severity of the psychopathology did not differentiate the two groups of children. Among factors related to the mental status of the children were: severity and onset under 30 years of age of the parental depression and lifelong association of parental anxiety with depression. Personality measurements performed in children showed different personality structures in proband offspring. Data on adolescent psychopathology and personality showed little evidence of a homotypic relationship with the adult affective disorders.

Reproductive ability of workmen occupationally exposed to lead.

The reproductive ability of 150 men occupationally exposed to lead were studied by clinical and toxicological analysis. Subjects were divided into four groups: lead-poisoned workmen (23) and those showing a moderate (42), slight (35), or physiologic absorption (50). Findings show that (1) Lead poisoning as well as moderate increased absorption of lead decrease the fertile ability of men. An increased frequency of asthenospermia, hypospermia, and teratospermia have objectified the decrease. (2) Slight increased or physiologic absorption of lead do not significantly influence the fertile ability of workmen. (3) Hypofertility induced by lead is due, perhaps, to its direct toxic effect on the gonads, as no interference with the hypothalamopituitary axis were evidenced.

Correlations between intratumoral vascular network and tumoral architecture in prostatic adenocarcinoma.

The authors made a preliminary assessment of possible correlations between the intratumoral vascular density (IVD) and the architectural tumoral patterns described by Gleason. The studied material consisted of samples obtained by transurethral resection from 34 patients diagnosed with prostatic adenocarcinoma. Ten fields, five for dominant and five for secondary identified patterns of each case, with no necrosis were selected randomly from CD34 immunomarked sections using ×20 objective. IVD increased with Gleason pattern both for the entire group, but also for "solid" phenotype group of subtypes up to pattern 4, respectively subtype 4B. In "necrotizing" phenotype group of subtypes, IVD had a decreasing trend from the better-differentiated subtypes to the poorest one. These preliminary data showed that the intratumoral vascular network reacts differently to the loss of tumoral differentiation in the two groups of Gleason subtypes suggesting the existence of two different populations of malignant cells.

Correlations between intratumoral interstitial fibrillary network and tumoral architecture in prostatic adenocarcinoma.

The authors made a preliminary assessment of possible correlations between the amount of intratumoral stromal fibrillary components (ISFC) and the architectural tumoral patterns described by Gleason. The studied material consisted of samples obtained by transurethral resection from 34 patients diagnosed with prostatic adenocarcinoma. Ten fields, five for dominant and five for secondary identified patterns of each case, with no necrosis were selected randomly from Gömöri stained sections using ×20 objective. ISFC-ratio increased with Gleason pattern both for the entire group but also for "Necrotizing" phenotype patterns and "Solid" phenotype patterns, excepting the subtype "4A" where the stromal compartment was reduced by the expansion of tumoral ducts enlarged by growing tumoral intraductal cribriform masses. These preliminary data showed that stromal microenvironment try to adapt to the loss of tumoral differentiation by increasing the amount of fibrillary components of intratumoral stromal compartment.

Replication of functional serotonin receptor type 3A and B variants in bipolar affective disorder: a European multicenter study.

Serotonin type 3 receptors (5-HT(3)) are involved in learning, cognition and emotion, and have been implicated in various psychiatric phenotypes. However, their contribution to the pathomechanism of these disorders remains elusive. Three single nucleotide polymorphisms (SNPs) in the HTR3A and HTR3B genes (rs1062613, rs1176744 and rs3831455) have been associated with bipolar affective disorder (BPAD) in pilot studies, and all of them are of functional relevance. We performed a European multicenter study to confirm previous results and provide further evidence for the relevance of these SNPs to the etiology of neuropsychiatric disorders. This involved analysis of the distribution of the three SNPs among 1804 BPAD cases and 2407 healthy controls. A meta-analysis revealed a pooled odds ratio of 0.881 (P = 0.009, 95% confidence intervals = 0.802-0.968) for the non-synonymous functional SNP HTR3B p.Y129S (rs1176744), thereby confirming previous findings. In line with this, the three genome-wide association study samples BOMA (Bonn-Mannheim)-BPAD, WTCCC (Wellcome Trust Case Control Consortium)-BPAD and GAIN (Genetic Association Information Network)-BPAD, including >3500 patients and 5200 controls in total, showed an overrepresentation of the p.Y129 in patients. Remarkably, the meta-analysis revealed a P-value of 0.048 (OR = 0.934, fixed effect model). We also performed expression analyses to gain further insights into the distribution of HTR3A and HTR3B mRNA in the human brain. HTR3A and HTR3B were detected in all investigated brain tissues with the exception of the cerebellum, and large differences in the A:B subunit ratio were observed. Interestingly, expression of the B subunit was most prominent in the brain stem, amygdalae and frontal cortex, regions of relevance to psychiatric disorders. In conclusion, the present study provides further evidence for the presence of impaired 5-HT(3) receptor function in BPAD.

Phylloides tumours of the breast. A clinicopathologic study of 19 cases.

During a period of 29 years (1960-1989), 19 patients with benign (15 cases) or malignant (4 cases) phylloides tumours were seen at the Surgical Clinic of Fundeni Hospital. Fourteen patients were followed up for a period of 2 to 21 years (mean 9.3 years). Two patients with benign tumours developed malignant recurrences. The malignant potential could not be predicted by histologic criteria. Although 4 patients had malignant tumours and other 2 developed malignant recurrences later, no distant metastases and no death in the 14 patients available for follow up were observed. We believe that in large or malignant tumours simple mastectomy is the surgical treatment of choice, in most benign phylloides tumours a conservative approach (quadrantectomy or lumpectomy) may be considered of the patients can be adaquately followed up.

A rating scale for the severity of psychopathology in children.

The Scale for Assessing Severity of the Psychopathology in Children (SSPC) is intended to measure the overall psychosocial impairment caused to children and adolescents by psychic disorders. The scale contains 3 items (number of impaired areas of psychosocial functioning and duration of the modifications of child's behaviour; decrease of school performances below the expected level according to IQ; subjective distress caused by the psychic disorder either to the child or to the family leading to call for medical help) defining a single dimension. Each item is scaled on four degrees of impairment and described behaviourally in a detailed way. The total score summed over the 3 items corresponds to four levels of severity of the psychopathology. Interrater reliability, sensitivity to change and concurrent validity of the SSPC are examined over three studies.

A trial to apply the concept of genomic imprinting to the manic-depressive illness.

The phenotypic indicators of genomic imprinting were applied to the familial psychopathology data collected through the family history method about 886 adult relatives of 65 manic-depressive probands directly investigated. The probands and their relatives were diagnosed according to DSM-III/DSM-III-R criteria. A first analysis of the age at onset of the BP illness by affective status of the probands' parents suggested that the BP disorder begins about 8 years earlier in the probands whose father was affectively ill (13.84% cases) than in the probands whose mother was affectively ill (24.6% cases) (t = -3.29, P < .004). When controlling this result for the effect of the probands' sex, its statistical significance decreased. The severity of the BP illness seemed also to be influenced by the affective status of the probands' father but only when assessing the probands' illness severity over a long time period and taking into account their psychosocial functioning; the number of manic and depressive hospitalized and non-hospitalized episodes as a single measure of the BP disorder severity as well as the morbidity risk in the first degree relatives of the probands did not significantly differentiate the patients whose disorder was transmitted by the father/paternal side as compared with the patients who inherited the BP disorder from the mother/maternal side.

Study of the risk factors for upper digestive hemorrhage, determined by post-mortem investigations in 39 cases of liver cirrhosis.

A retrospective study of the causes and the risk factors for upper digestive hemorrhage (UDH) was carried out by post-mortem investigations in 39 consecutive cases of liver cirrhosis (LC), in comparison with a control group of 40 patients with LC, free of UDH. The patients' age and the disease duration in the cases with ruptured esophageal varices or with hemorrhagic erosive gastritis (the main causes of UDH in the group studied) were longer than in the controls. The causes of UDH were: rupture of esophageal varices (in 43.7% of the cases), hemorrhagic erosive gastritis (41%), both (10.2%), and active duodenal ulcer (5.1%). The frequency of small esophageal varices among the ruptured ones (23%) was higher than that detected by endoscopic studies (13-15%), owing to the peculiarities of the group investigated and to the methods of evaluation. Ascites can be considered a risk factor for UDH, as an expression of portal hypertensions. Liver carcinoma, jaundice, prothrombinemia, albuminemia, bilirubinemia and Child index are not risk factors for UDH, some of them reflecting only a longer evolution of the disease.

[Postoperative intestinal infarction]. / Infarctul intestinal postoperator.

A total of 39 cases are analized, of post-operative intestinal infarction, that occured exclusively in patients with cardiovascular diseases. The clinical signs appeared early in most of the cases (85,4% in the first 72 hours; 48,4% in the first 24 hours) and the manifestations were typical in 42,4% and non-significant in 35% of the cases. Since none of the cases benefited from a successful therapy the authors suggest that stress should be laid on prophylactic measures (correct re-equillibration).