Detecting Impending Stroke From Cognitive Traits Evident in Internet Searches: Analysis of Archival Data.

BACKGROUND: Cerebrovascular disease is a leading cause of mortality and disability. Common risk assessment tools for stroke are based on the Framingham equation, which relies on traditional cardiovascular risk factors to predict an acute event in the near decade. However, no tools are currently available to predict a near/impending stroke, which might alert patients at risk to seek immediate preventive action (eg, anticoagulants for atrial fibrillation, control of hypertension). OBJECTIVE: Here, we propose that an algorithm based on internet search queries can identify people at increased risk for a near stroke event. METHODS: We analyzed queries submitted to the Bing search engine by 285 people who self-identified as having undergone a stroke event and 1195 controls with regard to attributes previously shown to reflect cognitive function. Controls included random people 60 years and above, or those of similar age who queried for one of nine control conditions. RESULTS: The model performed well against all comparator groups with an area under the receiver operating characteristic curve of 0.985 or higher and a true positive rate (at a 1% false-positive rate) above 80% for separating patients from each of the controls. The predictive power rose as the stroke date approached and if data were acquired beginning 120 days prior to the event. Good prediction accuracy was obtained for a prospective cohort of users collected 1 year later. The most predictive attributes of the model were associated with cognitive function, including the use of common queries, repetition of queries, appearance of spelling mistakes, and number of queries per session. CONCLUSIONS: The proposed algorithm offers a screening test for a near stroke event. After clinical validation, this algorithm may enable the administration of rapid preventive intervention. Moreover, it could be applied inexpensively, continuously, and on a large scale with the aim of reducing stroke events.

Improving blood glucose level predictability using machine learning.

This study was designed to improve blood glucose level predictability and future hypoglycemic and hyperglycemic event alerts through a novel patient-specific supervised-machine-learning (SML) analysis of glucose level based on a continuous-glucose-monitoring system (CGM) that needs no human intervention, and minimises false-positive alerts. The CGM data over 7 to 50 non-consecutive days from 11 type-1 diabetic patients aged 18 to 39 with a mean HbA1C of 7.5% ± 1.2% were analysed using four SML models. The algorithm was constructed to choose the best-fit model for each patient. Several statistical parameters were calculated to aggregate the magnitudes of the prediction errors. The personalised solutions provided by the algorithm were effective in predicting glucose levels 30 minutes after the last measurement. The average root-mean-square-error was 20.48 mg/dL and the average absolute-mean-error was 15.36 mg/dL when the best-fit model was selected for each patient. Using the best-fit-model, the true-positive-hypoglycemia-prediction-rate was 64%, whereas the false-positive- rate was 4.0%, and the false-negative-rate was 0.015%. Similar results were found even when only CGM samples below 70 were considered. The true-positive-hyperglycemia-prediction-rate was 61%. State-of-the-art SML tools are effective in predicting the glucose level values of patients with type-1diabetes and notifying these patients of future hypoglycemic and hyperglycemic events, thus improving glycemic control. The algorithm can be used to improve the calculation of the basal insulin rate and bolus insulin, and suitable for a closed loop "artificial pancreas" system. The algorithm provides a personalised medical solution that can successfully identify the best-fit method for each patient.

Pancreatic Beta-Cell Proliferation Induced by Estradiol-17ß is Foxo1 Dependent.

Estradiol-17ß (E2) and the Foxo1 transcription factor have each been implicated in the regulation of ß-cell proliferation. Interaction between Foxo1and estrogen receptor alpha (ERα), effecting cell cycle, has been demonstrated in breast cancer cells, but has not been studied thus far in ß-cells. Using human islets and the INS1-E ß-cell line, this study investigated the contribution of Foxo1 to E2-mediated ß-cell replication. Foxo1 expression was knocked down in INS1-E cells using siRNA and Foxo1 activity was inhibited in human islets with a specific Foxo1 inhibitor (AS1842856). Cells were treated with E2 and the ERα agonist PPT and evaluated for proliferation by 3[H]-thymidine incorporation and for transcriptional activity through the estrogen response element by the luciferase assay. As Foxo1 activity is regulated by post-translational modifications, the effect of E2 on phosphorylation was also assessed. In INS1-E cells, knock down of Foxo1 abrogated the proliferative response to E2 and PPT. In human islets, inhibition of Foxo1 abrogated E2-mediated proliferation and attenuated the response to PPT. Foxo1 knock down and inhibition reduced activity through the estrogen response element by 25% (p<0.05) and 50% (p<0.01) respectively, in INS1-E cells. E2 increased Foxo1 phosphorylation in a time dependent manner in INS1-E and human islets (p<0.01, p<0.05, respectively). These findings suggest that Foxo1 is involved in E2-mediated proliferation in INS1-E cells and human islets. This may have implications vis-à-vis variations in circulating endogenous E2 concentrations in diabetes.

The Proatherogenic Effect of Chronic Nitric Oxide Synthesis Inhibition in ApoE-Null Mice Is Dependent on the Presence of PPAR α.

Inhibition of endothelial nitric oxide synthase (eNOS) accelerates atherosclerosis in ApoE-null mice by impairing the balance between angiotensin II (AII) and NO. Our previous data suggested a role for PPAR α in the deleterious effect of the renin-angiotensin system (RAS). We tested the hypothesis that ApoE-null mice lacking PPAR α (DKO mice) would be resistant to the proatherogenic effect of NOS inhibition. DKO mice fed a Western diet were immune to the 23% worsening in aortic sinus plaque area seen in the ApoE-null animals under 12 weeks of NOS inhibition with a subpressor dose of L-NAME, P = 0.002. This was accompanied by a doubling of reactive oxygen species (ROS-) generating aortic NADPH oxidase activity (a target of AII, which paralleled Nox1 expression) and by a 10-fold excess of the proatherogenic iNOS, P < 0.01. L-NAME also caused a doubling of aortic renin and angiotensinogen mRNA level in the ApoE-null mice but not in the DKO, and it upregulated eNOS in the DKO mice only. These data suggest that, in the ApoE-null mouse, PPAR α contributes to the proatherogenic effect of unopposed RAS/AII action induced by L-NAME, an effect which is associated with Nox1 and iNOS induction, and is independent of blood pressure and serum lipids.

Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2ß in the human osteosarcoma cell line U2OS.

BACKGROUND: Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2ß plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2ß in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. RESULTS: By knocking down the expression of LAP2ß, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2ß knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2ß knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2ß knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. CONCLUSIONS: Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2ß reduced expression. Our study implies on a novel role of LAP2ß in the maintenance of cell ploidy status. LAP2ß depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.

LAP2zeta binds BAF and suppresses LAP2beta-mediated transcriptional repression.

Proteins of the nuclear envelope have been implicated as participating in gene silencing. BAF, a DNA- and LEM domain-binding protein, has been suggested to link chromatin to the nuclear envelope. We have previously shown that LAP2beta, a LEM-domain inner nuclear membrane protein, represses transcription through binding to HDAC3 and induction of histone H4 deacetylation. We now show that LAP2zeta, the smallest LAP2 family member, is also involved in regulation of transcription. We show that similar to other LEM-domain proteins LAP2zeta interacts with BAF. LAP2zeta-YFP and BAF co-localize in the cytoplasm, and overexpression of LAP2zeta leads to reduction of nucleoplasmic BAF. Mutations in the LAP2zeta-YFP LEM domain decrease its interaction with BAF retaining the nucleo-cytoplasmic distribution of BAF. Co-expression of LAP2beta and LAP2zeta results in inhibition of LAP2beta-induced gene silencing while overexpression of LAP2zeta alone leads to a small increase in transcriptional activity of various transcription factors. Our results suggest that LAP2zeta is a transcriptional regulator acting predominantly to inhibit LAP2beta-mediated repression. LAP2zeta may function by decreasing availability of BAF. These findings could have implications in the study of nuclear lamina-associated diseases and BAF-dependent retroviral integration.

FreeStyle Mini blood glucose results are accurate and suitable for use in glycemic clamp protocols.

OBJECTIVE: We assessed the accuracy of the FreeStyle Mini (FSM) meter for use in glycemic clamp and meal protocols in comparison with the HemoCue Glucose 201 DM Analyzer (HemoCue) and the YSI 2300 STAT Glucose Oxidase Analyzer (YSI). METHODS: Seven volunteers with type 2 diabetes mellitus, 35-69 years old, underwent a frequently sampled meal test and a graded hyperglycemic test, on two separate days, with one of the volunteers undergoing each test twice. Samples for glucose measurements were obtained from arterialized venous blood. A total of 420 samples (with glucose levels ranging from 63 to 388 mg/dl) were available for comparison. On average, 10 measurements were available for every 5 mg/dl increment in glucose level in the range of 130-310 mg/dl. Blood glucose measurements were done on each sample with the FSM, HemoCue, and YSI. RESULTS: FreeStyle Mini blood glucose values correlated closely with the YSI readings. Of the FSM measurements, 99.0% were within the Clarke error grid zone A; 51.3%, 84.7%, and 96.2% of the FSM readings were within 5%, 10% and 15% of the YSI values, respectively. The FSM was significantly more accurate than the HemoCue (84.7% vs 76.6% of results within 10% of the YSI results; p = .0038). The mean average relative difference of the FSM (5.8%) was also significantly lower than that of the HemoCue (6.8%; p = .0013) CONCLUSIONS: The FSM provides accurate results and constitutes a suitable alternative for bedside blood glucose measurements in experimental procedures, helping to reduce sample size, turnaround time, and cost.

Gene silencing at the nuclear periphery.

The nuclear envelope (NE) is composed of inner and outer nuclear membranes (INM and ONM, respectively), nuclear pore complexes and an underlying mesh like supportive structure--the lamina. It has long been known that heterochromatin clusters at the nuclear periphery adjacent to the nuclear lamina, hinting that proteins of the lamina may participate in regulation of gene expression. Recent studies on the molecular mechanisms involved show that proteins of the nuclear envelope participate in regulation of transcription on several levels, from direct binding to transcription factors to induction of epigenetic histone modifications. Three INM proteins; lamin B receptor, lamina-associated polypeptide 2beta and emerin, were shown to bind chromatin modifiers and/or transcriptional repressors inducing, at least in one case, histone deacetylation. Emerin and another INM protein, MAN1, have been linked to down-regulation of specific signaling pathways, the retino blastoma 1/E2F MyoD and transforming growth factor beta/bone morphogenic protein, respectively. Therefore, cumulative data suggests that proteins of the nuclear lamina regulate transcription by recruiting chromatin modifiers and transcription factors to the nuclear periphery. In this minireview we describe the recent literature concerning mechanisms of gene repression by proteins of the NE and suggest the hypothesis that the epigenetic "histone code", dictating transcriptional repression, is "written" in part, at the NE by its proteins. Finally, as aberrant gene expression is one of the mechanisms speculated to underlie the newly discovered group of genetic diseases termed nuclear envelopathies/laminopathies, elucidating the repressive role of NE proteins is a major challenge to both researchers and clinicians.

Enhanced expression of the nuclear envelope LAP2 transcriptional repressors in normal and malignant activated lymphocytes.

Extensive research in recent years has broadened the functions of nuclear envelope proteins beyond simply stabilizing the nucleus architecture. Particularly, integral nuclear membrane proteins, such as the alternative spliced isoforms of lamina-associated polypeptide 2 (LAP2), have been shown to be important for the initiation of replication and repression of transcription. The latter is regulated by epigenetic changes, induced by the binding of LAP2beta to histone deacetylase-3 (HDAC3), resulting in histone H4 deacetylation. Involvement of nuclear envelope proteins in pathological proliferative conditions, mainly those involving abnormal recruitment and activation of HDACs, is still unknown. In this paper, we show that various nuclear envelope proteins are highly expressed in normal and malignant activated lymphocytes. Specifically, rapidly replicating cells of various hematological malignancies highly express LAP2beta, while slowly proliferating malignant cells of chronic malignant hematological diseases do not. Taking together the elevated expression of LAP2beta in highly proliferative malignant cells with its known ability to modify histones through binding with HDAC3 raises the possibility of its role in hematological malignancies involving aberrant activity of HDAC3. Based on our presented results, we believe that the LAP2-HDAC regulatory pathway should be studied as a new target for rational therapy.

BCL6 is regulated by p53 through a response element frequently disrupted in B-cell non-Hodgkin lymphoma.

The BCL6 transcriptional repressor mediates survival, proliferation, and differentiation blockade of B cells during the germinal-center reaction and is frequently misregulated in B-cell non-Hodgkin lymphoma (BNHL). The p53 tumor-suppressor gene is central to tumorigenesis. Microarray analysis identified BCL6 as a primary target of p53. The BCL6 intron 1 contains a region in which 3 types of genetic alterations are frequent in BNHL: chromosomal translocations, point mutations, and internal deletions. We therefore defined it as TMDR (translocations, mutations, and deletions region). The BCL6 gene contains a p53 response element (p53RE) residing within the TMDR. This p53RE contains a motif known to be preferentially targeted by somatic hypermutation. This p53RE is evolutionarily conserved only in primates. The p53 protein binds to this RE in vitro and in vivo. Reporter assays revealed that the BCL6 p53RE can confer p53-dependent transcriptional activation. BCL6 mRNA and protein levels increased after chemotherapy/radiotherapy in human but not in murine tissues. The increase in BCL6 mRNA levels was attenuated by the p53 inhibitor PFT-alpha. Thus, we define the BCL6 gene as a new p53 target, regulated through a RE frequently disrupted in BNHL.

The nuclear-envelope protein and transcriptional repressor LAP2beta interacts with HDAC3 at the nuclear periphery, and induces histone H4 deacetylation.

Nuclear-envelope proteins have been implicated in diverse and fundamental cell functions, among them transcriptional regulation. Gene expression at the territory of the nuclear periphery is known to be repressed by epigenetic modifications such as histone deacetylation and methylation. However, the mechanism by which nuclear-envelope proteins are involved in such modifications is still obscure. We have previously shown that LAP2beta, an integral nuclear-envelope protein that contains the chromatin-binding LEM domain, was able to repress the transcriptional activity of the E2F5-DP3 heterodimer. Here, we show that LAP2beta's repressive activity is more general, encompassing various E2F members as well as other transcription factors such as p53 and NF-kappaB. We further show that LAP2beta interacts at the nuclear envelope with HDAC3, a class-I histone deacetylase, and that TSA (an HDAC inhibitor) abrogates LAP2beta's repressive activity. Finally, we show that LAP2beta is capable of inducing histone-H4 deacetylation. Our data provide evidence for the existence of a previously unknown repressive complex, composed of an integral nuclear membrane protein and a histone modifier, at the nuclear periphery.

Nuclear envelopathies--raising the nuclear veil.

The nuclear envelope separates the chromosomes from cytoplasm in eukaryotic cells and consists of three main domains: inner and outer nuclear membranes and nuclear pore complexes. The inner nuclear membrane maintains close associations with the underlying chromatin and nuclear lamina. For many years, the nuclear envelope was thought to function mainly as an architectural stabilizer of the nucleus, participating in assembly and disassembly processes during mitosis. However, recent findings demonstrate that nuclear envelope proteins are involved in fundamental nuclear functions, such as gene transcription and DNA replication, and that inherited or de novo mutated proteins cause human diseases, termed "nuclear envelopathies." These findings emphasize the importance of understanding the functions of this cellular domain, in both physiologic and pathologic states. To date, mutations in the genes encoding the nuclear envelope proteins emerin, MAN1, lamin A/C, and lamin B receptor were found to cause nuclear envelopathies. The diseases that are caused by mutations in LMNA gene are collectively called "laminopathies." Nuclear envelopathies have diverse clinical phenotypes, ranging from cardiac and skeletal myopathies to partial lipodystrophy, peripheral neuropathy, and premature aging. This raises the question of how do such ubiquitously expressed proteins give rise to tissue-specific disease phenotypes. One possible explanation is the involvement of nuclear envelope proteins in the regulation of gene transcription, a novel mechanism that has been the focus of research in our lab in recent years. In this review, we describe recent discoveries in the field of nuclear envelopathies and discuss current proposed pathophysiological mechanisms underlying these diseases.