Hypoperfusion intensity ratio correlates with collaterals and predicts outcome and infarct volume in acute ischemic stroke patients.

BACKGROUND: Hypoperfusion Intensity Ratio (HIR) is associated with collaterals and outcome in acute ischemic stroke (AIS). We investigated whether a combined assessment of HIR and collaterals could provide an added value. METHODS: Retrospective single-center study, including AIS patients with large vessel occlusion and endovascular treatment 0-24 h from onset. Predictors of FIV and outcome (90 days modified Rankin Scale 0-1) were investigated with linear and logistic regression respectively. Subjects were stratified in three groups: poor collaterals (grade 0-3) with poor HIR (≥.4), good collaterals (grade 4-5) with poor HIR/poor collaterals with good HIR (<.4) and good collaterals with good HIR. RESULTS: We included 337 patients (median age 77, 53.1% males), of whom 100 (29.7%) had excellent outcome. One hundred and forty five patients with favourable collateral and HIR profiles had smaller infarct (median poor collaterals with poor HIR 41 mL, good collaterals with poor HIR/poor collaterals with good HIR 21 mL and good collaterals with good HIR 11 mL, p <.001) and higher rates of excellent outcome (poor collaterals with poor HIR 15.7%, good collaterals with poor HIR/poor collaterals with good HIR 26.2% and good collaterals with good HIR 39.3% p =.001). Logistic regression showed that patients with favourable collateral and HIR profiles had the highest odds of good outcome (OR: 3.83, 95% CI 1.62-9.08, p =.002). CONCLUSION: Collaterals and HIR are independent predictors of final infarct lesion and outcome in stroke patients and their integration provides an added value. These findings might inform clinical practice and future trials.

Pivotal role of multiphase computed tomography angiography for collateral assessment in patients with acute ischemic stroke.

The cerebral collateral circulation is the main compensatory mechanism that maintains the ischemic penumbra viable, the tissue at risk for infarction that can be saved if blood flow is restored by reperfusion therapies. In clinical practice, the extent of collateral vessels recruited after vessel occlusion can be easily assessed with computed tomography angiography (CTA) using two different techniques: single-phase CTA (sCTA) and multi-phase CTA (mCTA). Both these methodologies have demonstrated a high prognostic predictive value for prognosis due to the strong association between the presence of good collaterals and favorable radiological and clinical outcomes in patients with acute ischemic stroke (AIS). However, mCTA seems to be superior to sCTA in the evaluation of collaterals and a promising tool for identifying AIS patients who can benefit from reperfusion therapies. In particular, it has recently been proposed the use of mCTA eligibility criteria has been recently proposed for the selection of AIS patients suitable for endovascular treatment instead of the current accepted criteria based on CT perfusion. In this review, we analyzed the characteristics, advantages and disadvantages of sCTA and mCTA to better understand their fields of application and the potential of mCTA in becoming the method of choice to assess collateral extent in AIS patients.

Association between perihematomal perfusion and intracerebral hemorrhage shape.

PURPOSE: The pathophysiological determinants of irregular intracerebral hemorrhage (ICH) shape are unclear. We aimed at characterizing the relationship between perihematomal perfusion and ICH shape. METHODS: A single-center cohort of patients with primary ICH was analyzed. Patients underwent computed tomography perfusion within 6 h from onset. Cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) were calculated in the manually outlined perihematomal low-density region. ICH shape was rated on baseline non-contrast CT following international consensus criteria, and predictors of irregular shape were explored with logistic regression. RESULTS: A total of 150 patients were included, of whom 66 (44%) had irregular shape. Perihematomal CBF was lower in irregular ICH (median 23 vs 35 mL/100 g/min, p<0.001). CBF<20 mL/100 g/min was independently associated with irregular shape (odds ratio 9.67, 95% CI 2.42-38.69, p=0.001). CONCLUSION: Our findings suggest that perihematomal hypoperfusion may contribute to the CT appearance of acute ICH.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Systematic Review of Zinc's Benefits and Biological Effects on Oral Health.

BACKGROUND: This review was based on the following question: "What is the state-of-the-art regarding the effect of zinc exposure in the oral cavity on a population of adults and children, compared to dental products containing materials other than zinc, considering in vivo (clinical trials and observational studies) and in vitro studies?" according to a PICOS strategy format. This study aims to analyze zinc application in dental materials, with different compositions and chemical formulations, considering how mechanical and biological properties may influence its clinical applicability. METHODS: In vivo (clinical trials: controlled clinical trials (CCTs) and randomized controlled trials (RCTs); and observational studies: case control and cohort studies) trials or in vitro studies published in English or Italian during the last 10 years on children and adult patients with zinc exposure were included by three different reviewers using the MEDLINE (via PubMed), Scopus, and Web of Science electronic databases. RESULTS: Titles and abstracts were evaluated following the eligibility criteria. The full texts of eligible studies were then reviewed against the inclusion/exclusion criteria. Scientific and technical information of the 33 included studies were collected into evidence tables, reporting data on in vivo and in vitro studies. A narrative approach was adopted. CONCLUSIONS: Antibacterial activity was found to be the most studied property of zinc, but further investigations are needed to establish adjuvant zinc therapies in patients with oral disease.

Human DNA polymerase beta mutations allowing efficient abasic site bypass.

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase ß when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase ß was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase ß and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase ß variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.

Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis.

BACKGROUND: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. METHODS: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. RESULTS: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. CONCLUSIONS: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.

Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I.

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.

Synthesis of DNA strands site-specifically damaged by c8-arylamine purine adducts and effects on various DNA polymerases.

C8-Arylamine-dG and C8-arylamine-dA adducts have been prepared using palladium cross-coupling chemistry. These adducts were subsequently converted into the corresponding 5'-O-DMTr-C8-arylamine-3'-O-phosphoramidites and then used for the automated synthesis of different site-specifically modified oligonucleotides. These "damaged" oligonucleotides have been characterized by ESI-MS, UV thermal stability assays, and circular dichroism, and they have been used in EcoRI assays as well as in primer extension studies using various DNA polymerases.

Nucleotide insertion and bypass synthesis of pyrene- and BODIPY-modified oligonucleotides by DNA polymerases.

The chromophores pyrene and bordipyrromethenylbenzene directly linked to the 5-position of uridine are tolerated and recognized as thymine derivatives by DNA polymerases in primer extension experiments.

Psychological, cognitive and maternal stress assessment in children with primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare disorder due to structure and functional abnormalities of respiratory cilia. There are no reports on the behavioral and psychological aspects of children and adolescents with PCD. This study was undertaken to assess the cognitive and behavioural characteristics, and the parental stress of a population of school-aged children with PCD. METHODS: Ten PCD and 34 healthy school-aged children underwent Wechsler Intelligence Scale for Children-III edition, Child Behavior Check-List questionnaire (CBCL), Parenting Stress Index-Short Form tests in order to perform a behavioural and psychological evaluation. RESULTS: PCD children showed significant behavioral and social competent problems in CBCL scale than control children, in particular with regard to internalizing problems score (P<0.001). Parental distress, parent-child interaction and total stress in the mothers of PCD patients were higher than those in the controls' parents (P<0.001). CONCLUSION: Our findings pinpoint the importance of specific psychological support in the clinical management of children with PCD.

Low glycemic index diet in children and young adults with refractory epilepsy: first Italian experience.

This is the report on the first Italian experience with the low glycemic index diet (LGIT) in a group of children, adolescents and young adults with refractory epileptic encephalopathies. A retrospective chart review was performed on patients initiating the LGIT in an outpatient setting from 2005 to 2010. Demographic and clinical information including seizure type, baseline seizure frequency, medications, blood chemistry, side effects, and anthropometrics were collected. Patients were educated and followed by a dietician to restrict foods with high glycemic index and to limit total daily carbohydrates to 40-60g. Change in seizure frequency was assessed at each 3-month follow-up intervals in the first year and then at each 6-month intervals. Fifteen consecutive patients (13 males and 2 females, aged between 11.3 years and 22 years), almost all affected by generalized cryptogenic or symptomatic refractory epilepsy, were enrolled in the study. After a mean follow-up period of 14.5±6.5 months (median 12.0; range 1-60 months), 6 patients (40%) had a 75-90% seizure reduction, while seizures decreased by 50% in other 2 (13.3%) and were unchanged in 7 (46.7%). The diet was discontinued in 4 patients within the first 5 months. No adverse events occurred during the diet. In conclusion, this initial experience confirms that some refractory patients may improve on the LGIT, even as first dietary option.

Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y(12)-receptor.

The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC(50) concentrations of about 0.3nM. In cells expressing R256A constructs, the IC(50) concentration amounted to 25nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent pK(B)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y(12)-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta.

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.