Ultrafast electron dynamics in excited states of conjugated thiophene-fluorene organic polymer (pF8T2) thin films.

The electronic states of poly(9,9-dioctylfluorenyl-alt-bithiophene) pF8T2 on H/Si(100) substrates, prototypical for organic photovoltaics, were investigated by ultrafast photoelectron spectroscopy and by time-resolved fluorescence studies. Occupied and unoccupied electronic states were analysed by ultraviolet photoelectron spectroscopy (UPS), static and dynamic femtosecond two-photon photoemission (2PPE), and time-correlated single photon counting (TCSPC). Time-resolved measurements allow assessment of population lifetimes of intermediate states. The combination of time-resolved photoelectron spectroscopy and fluorescence excitation allows following the electronic dynamics in excited states from the femtosecond to the nanosecond time scale. For this prototypical material the electron kinetic energy resolved lifetimes range from about a few tens of femtoseconds up to hundreds of picoseconds. After annealing these types of organic thin films the efficiency of organic solar cells usually increases. We show that annealing does not influence the initial ultrafast charge generation processes, but the long-lived states. However, the nanosecond scale fluorescence lifetimes measured by TCSPC are prolonged after annealing, which therefore is identified as the cause of a greater exciton diffusion range and thus is beneficial for charge carrier extraction.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

Functional and radiological outcomes after bridging therapy versus direct thrombectomy in stroke patients with unknown onset: Bridging therapy versus direct thrombectomy in unknown onset stroke patients with 10-point ASPECTS.

BACKGROUND AND PURPOSE: The aim was to assess functional and radiological outcomes after bridging therapy (intravenous thrombolysis plus mechanical thrombectomy) versus direct mechanical thrombectomy (MT) in unknown onset stroke patients. METHODS: A cohort study was conducted on prospectively collected data from unknown onset stroke patients who received endovascular procedures at ≤6 h from symptom recognition or awakening time. RESULTS: Of the 349 patients with a 10-point Alberta Stroke Program Early Computed Tomography Score (ASPECTS), 248 received bridging and 101 received direct MT. Of the 134 patients with 6-9-point ASPECTS, 123 received bridging and 111 received direct MT. Each patient treated with bridging was propensity score matched with a patient treated with direct MT for age, sex, study period, pre-stroke disability, stroke severity, type of stroke onset, symptom recognition to groin time (or awakening to groin time), ASPECTS and procedure time. In the two matched groups with 10-point ASPECTS (n = 73 vs. n = 73), bridging was associated with higher rates of excellent outcome (46.6% vs. 28.8%; odds ratio 2.302, 95% confidence interval 1.010-5.244) and successful recanalization (83.6% vs. 63%; odds ratio 3.028, 95% confidence interval 1.369-6.693) compared with direct MT; no significant association was found between bridging and direct MT with regard to rate of symptomatic intracerebral hemorrhage (0% vs. 1.4%). In the two matched groups with 6-9-point ASPECTS (n = 45 vs. n = 45), no significant associations were found between bridging and direct MT with regard to rates of excellent functional outcome (44.4% vs. 31.1%), successful recanalization (73.3% vs. 76.5%) and symptomatic intracerebral hemorrhage (0% vs. 0%). CONCLUSIONS: Bridging at ≤ 6 h of symptom recognition or awakening time was associated with better functional and radiological outcomes in unknown onset stroke patients with 10-point ASPECTS.

Thrombolysis and bridging therapy in patients with acute ischaemic stroke and Covid-19.

BACKGROUND AND PURPOSE: Comorbidity of acute ischaemic stroke with Covid-19 is a challenging condition, potentially influencing the decision of whether to administer intravenous thrombolysis (IVT). We aimed to assess the 1-month outcome in ischaemic stroke patients with Covid-19 infection who received IVT alone or before thrombectomy (bridging therapy). METHODS: As a collaboration initiative promoted by the Italian Stroke Organization, all Italian stroke units (n = 190) were contacted and invited to participate in data collection on stroke patients with Covid-19 who received IVT. RESULTS: Seventy-five invited centers agreed to participate. Thirty patients received IVT alone and 17 received bridging therapy between 21 February 2020 and 30 April 2020 in 20 centers (n = 18, Northern Italy; n = 2, Central Italy). At 1 month, 14 (30.4%) patients died and 20 (62.5%) survivors had a modified Rankin Scale (mRS) score of 3 to 5. At 24 to 36 hours, asymptomatic intracerebral hemorrhage (ICH) was reported in eight (17.4%) patients and symptomatic ICH (sICH) in two (4.3%) patients. Causes of death were severe ischaemic stroke (n = 8), a new ischaemic stroke (n = 2), acute respiratory failure (n = 1), acute renal failure (n = 1), acute myocardial infarction (n = 1), and endocarditis (n = 1). In survivors with a 1-month mRS score of 3 to 5, baseline glucose level was higher, whereas endovascular procedure time in cases of bridging therapy was longer. Baseline National Institutes of Health Stroke Scale glucose and creatinine levels were higher in patients who died. CONCLUSIONS: Intravenous thrombolysis for patients with stroke and Covid-19 was not a rare event in the most affected areas by pandemic, and rates of 1-month unfavorable outcomes were high compared to previous data from the pre-Covid-19 literature. However, risk of sICH was not increased.

Fatal intracranial haemorrhage occurring after oral anticoagulant treatment initiation for secondary stroke prevention in patients with atrial fibrillation.

BACKGROUND AND PURPOSE: In this pooled analysis of seven multicentre cohorts potential differences were investigated in the incidence, characteristics and outcomes between intracranial haemorrhages (ICHs) associated with the use of non-vitamin K antagonist oral anticoagulants (NOAC-ICH) or with vitamin K antagonists (VKA-ICH) in ischaemic stroke patients after oral anticoagulant treatment initiation for atrial fibrillation (AF). METHODS: Data from 4912 eligible AF patients who were admitted in a stroke unit with ischaemic stroke or transient ischaemic attack and who were treated with either VKAs or NOACs within 3 months post-stroke were included. Fatal ICH was defined as death occurring during the first 30 days after ICH onset. A meta-analysis of available observational studies reporting 30-day mortality rates from NOAC-ICH or VKA-ICH onset was additionally performed. RESULTS: During 5970 patient-years of follow-up 71 participants had an ICH, of whom 20 were NOAC-ICH and 51 VKA-ICH. Patients in the two groups had comparable baseline characteristics, except for the higher prevalence of kidney disease in VKA-ICH patients. There was a non-significant higher number of fatal ICH in patients with VKAs (11 events per 3385 patient-years) than in those with NOACs (three events per 2623 patient-years; hazard ratio 0.32, 95% confidence interval 0.09-1.14). Three-month functional outcomes were similar (P > 0.2) in the two groups. The meta-analysis showed a lower 30-day mortality risk for patients with NOAC-ICH compared to VKA-ICH (relative risk 0.70, 95% confidence interval 0.51-0.95). CONCLUSIONS: Non-vitamin K oral anticoagulants for intracranial haemorrhages and VKA-ICH occurring during secondary stroke prevention of AF patients have comparable baseline characteristics and outcomes except for the risk of fatal ICH within 30 days, which might be greater in VKA-ICH.

Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways.

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells.

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.

Hemi- and monoataxia in cerebellar hemispheres and peduncles stroke lesions: topographical correlations.

Limb ataxia of sudden onset is due to a vascular lesion in either the cerebellum or the brainstem (posterior circulation, PC, territory). This sign can involve both the upper and the lower limb (hemiataxia) or only one limb (monoataxia). The topographical correlates of limb ataxia have been studied only in brainstem strokes. Therefore, it is not yet known whether this sign is useful to localize the lesion within the entire cerebellar system, both the cerebellar hemisphere and the cerebellar brainstem pathways. Limb ataxia was semi-quantified according to the International Cooperative Ataxia Rating Scale in 92 consecutive patients with acute PC stroke. Limb ataxia was present in 70 patients. Four topographical patterns based on magnetic resonance imaging findings were identified: picaCH pattern (posterior inferior cerebellar artery infarct); scaCH pattern (superior cerebellar artery infarct); CH/CP pattern (infarct involving both the cerebellum and the brainstem cerebellar pathways); and CP pattern (infarct involving the brainstem cerebellar pathways). Hemiataxia was present in (47/70; 67.1%) and monoataxia in (23/70; 32.9%) of patients. Monoataxia involved the upper limb in (19/70; 27.1%) and the lower limb in (4/70; 5.7%) of patients. Limb ataxia usually localized the lesion ipsilaterally (picaCH, scaCH, CH/CP, and CP patterns involving the medulla and sometimes the pons) (53/70; 75.7%), but it might be due also to contralateral (CP pattern involving the pons or midbrain) (16/70; 22.9%) or bilateral lesions (1/70). Limb ataxia usually localizes the lesion ipsilaterally but the infarct might be sometimes contralateral. The occurrence of monoataxia may suggest that the cerebellar system is somatotopically organized.

Alternative splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn.

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.