text:
“Cara’s clarity, rigour, directness, honesty, persistence and evident interest in the work she undertakes is the thing I enjoy the most about working with her.
I would recommend Cara without hesitation, and I already have! Cara makes sure to get her head around what’s really needed early on in a project, and she’s not afraid to ask difficult questions, she’s timely and always highly professional. Cara genuinely lives up to her pocketknife identity – she has a broad range of very high quality skills.”
“Cara has been essential and invaluable in helping me move forward with my business. Her attention to detail, commitment and knowledge of how my business works never fails to amaze me and it gives me the confidence and support to move forward.”
“I have worked with Cara for a number of years and I can honestly say, the work she did for me turned my business around.
The two main areas I would highlight where she adds incredible value are:
1. Looking at current process, and then working out what processes are required by teasing that information out of you.
She then goes ahead to implement a robust process and documentation to back this up.
2. “Out of the box” thinking on generating new business.
The second point was where I saw a 120% growth in New clients and over 100% increase in revenue from one simple idea. This actually saved my business and would not have been possible without Cara working with me, and really understanding my business and how it works and then coming up with an idea that really engaged my audience.“
“Cara has helped me so much! I’m a freelance Arts Practitioner and I had never given the time or commitment to running a business, I was always just jumping from one thing to the next. Cara took the time to help me consider my own business as a creative entity and take away all the fear I had about approaching ‘business’ stuff. I can’t thank her enough!”
Sarah, Arts Practitioner
“Cara has a creative mind but also is meticulous when developing process and documentation. Her passion to “do good” is seen through everything she does, and she genuinely cares about helping.
She knows how to question, and push you to get the very best out of you, and makes this an enjoyable experience.
I would highly recommend Cara to any business, but in particular, the smaller start up’s where you need someone who can produce a good background process for your business, come up with creative ideas for new business, maybe assist with websites, as she is a whizz on word press and creative writing.
Basically, she can throw her hand to a lot of things, but she can take an idea and represent it in a unique way, and that is a rare quality. Thank you Cara, for everything you have done and continue to do.”


text:
Couples Rehab for Addiction Recovery
Welcome to Denton, Montana – a serene and picturesque city nestled in the heart of the United States. Known for its breathtaking landscapes and tranquil atmosphere, Denton provides the perfect backdrop for couples seeking addiction treatment and recovery. With a range of specialized programs and intensive couples therapy, Denton offers a haven for couples looking to heal and rebuild their lives together.
Couples Addiction Treatment
When it comes to addiction, seeking treatment as a couple can be a powerful and effective approach. Couples addiction treatment programs in Denton recognize the unique dynamics and challenges that couples face when dealing with substance abuse. These programs aim to provide comprehensive care that addresses both individual and relationship needs.
Through a combination of individual therapy, group counseling, and joint sessions, couples addiction treatment programs help couples develop healthier coping mechanisms, improve communication, and rebuild trust. These programs also equip couples with the necessary tools and strategies to support each other’s recovery journey.
Intensive Couples Therapy
Intensive couples therapy is a key component of rehab for couples in Denton. This specialized form of therapy focuses on addressing the underlying issues that contribute to addiction and relationship challenges. Through intensive counseling sessions, couples gain a deeper understanding of their individual struggles and how they impact their relationship.
With the guidance of experienced therapists, couples learn effective communication techniques, conflict resolution skills, and strategies to rebuild trust and intimacy. Intensive couples therapy provides a safe and supportive environment where couples can openly explore their emotions, heal past wounds, and strengthen their bond.
Rehab for Couples
Rehab for couples in Denton offers a comprehensive range of treatment options tailored to meet the unique needs of couples struggling with substance abuse. These programs provide a structured and supportive environment where couples can embark on their journey to recovery together.
From detoxification and medical care to individual and group therapy, rehab for couples offers a holistic approach to addiction treatment. Couples engage in activities and workshops that promote self-reflection, personal growth, and relationship building. With the guidance of experienced addiction specialists, couples develop the necessary skills and strategies to maintain sobriety and build a healthier future together.
Substance Abuse Treatment for Couples
Substance abuse treatment for couples in Denton is designed to address the unique challenges faced by couples struggling with addiction. These programs provide a supportive and non-judgmental environment where couples can work together towards recovery.
Substance abuse treatment for couples focuses on helping couples identify and address the underlying causes of their addiction. Through individual and joint therapy sessions, couples develop healthier coping mechanisms, improve communication, and rebuild their lives free from substance abuse. These programs also provide education and support for relapse prevention, ensuring couples have the tools and resources to maintain long-term sobriety.
Couples Rehab Near Me
Denton, Montana, offers a sanctuary for couples seeking addiction treatment and recovery. With its serene surroundings and comprehensive treatment options, Denton provides a supportive and healing environment for couples to rebuild their lives together. Through couples addiction treatment, intensive therapy, and substance abuse treatment, couples in Denton can embark on a transformative journey towards healing and a brighter future.
Discover the transformative power of couples rehab in Denton, Montana. Learn about the comprehensive addiction treatment options, intensive couples therapy, and substance abuse treatment available for couples seeking recovery and healing.

domain: sociology


text:
If you give a mouse a beer, he’s going to ask for a cookie—and another, and another. If you give a person enough beer, she might find herself wolfing down a plate of greasy nachos. But why does binge drinking make us binge eat as well? The reason may lie not in the stomach but in the brain, recent research suggests. A study published today in Nature Communications found alcohol activated brain cells that control hunger, sending drunk mice scampering for snacks even when they were not really hungry.
Researchers from The Francis Crick Institute Mill Hill Laboratory in London got mice drunk, then tagged and recorded the electrical activity in brain cells linked to hunger, uncovering a neural mechanism that could explain why the animals ate significantly more after binge-drinking sessions even though their bodies did not need the calories.
Although hunger pangs in our stomach usually alert us that it is time to eat, the impulse to consume food originates in our brains, and brain cells located in the hypothalamus called agouti-related protein (AgRP) neurons play a key role in controlling hunger. A previous study showed that when AgRP neurons are activated, mice almost immediately seek out food and start eating, even if their stomachs are full. By contrast, when AgRP neurons are deactivated, hungry mice will not eat. AgRP neurons play a similar role in human hunger: Under natural conditions they are activated when our bodies need calories, signaling to us that we should find food. Something different happens, however, when alcohol is involved. Although alcohol is second only to fat in caloric density, previous studies have shown drinking causes humans to eat more, a paradox that made lead authors Craig Blomeley and Sarah Cains and colleagues wonder whether the brain could be to blame.
The neuroscientists first set out to confirm that consuming alcohol compels mice to eat more. They injected alcohol into the abdomens of male and female mice (housed alone to control for social factors) once a day for three days, mimicking an “alcohol weekend.” Each injection was the mouse-equivalent of roughly two bottles of wine, or six to eight British pints—what Blomeley calls “a proper binge session.” Over the three-day period intoxicated mice of both sexes ate significantly more than sober mice—a difference that was particularly pronounced on day two of the binge.
To investigate whether AgRP neurons are partly responsible for this behavior, the researchers sliced mouse brains into thin sections and tagged the AgRP neurons (kept alive with a nutrient solution) so calcium in the cells would glow green under a microscope—a proxy for cell activity. When the researchers bathed the sections in different concentrations of alcohol, they found that more alcohol caused calcium levels to increase. Next, the researchers exposed the neurons to different concentrations of alcohol and used probes to record the cells’ electrical activity. They found that alcohol exposure alters calcium exchange in the cells, which causes AgRP neurons to fire more frequently and easily. Activity in AgRP neurons returned to normal after the alcohol was flushed away, indicating the cells were not permanently altered.
Together, these results suggest alcohol directly activates AgRP neurons in the brain, which is what keeps mice munching away even when they do not need to eat. But what does this tell us about about our own alcohol-related eating habits? Blomeley and Cains point out that whereas we cannot definitely say whether the mechanism works the same way in humans, this brain circuitry is evolutionarily ancient and has been highly conserved across mammal species. “I don't doubt that AgRP neurons are activated in humans,” Cains says, “and that's why you see this effect.” Understanding the relationship between alcohol consumption and overeating is particularly important within the context of obesity, she adds. Although obesity is on the rise in many developed countries and alcohol intake has also increased, the two are often studied separately. “This study ties them together,” she says. The work “shows that if you have an increased alcohol intake, then you're going to, as a result of that through the effect of alcohol on your brain, have an elevated level of food intake.”
Scott Sternson, a neuroscientist at Howard Hughes Medical Institute who was not involved in the research, says the work is the first to show how alcohol activates AgRP neurons. He considers the study “an interesting and unexpected starting point” for further research, but questions whether the high alcohol levels used in the study are consistent with the kind of drinking often associated with overeating in humans. Compared with that of mice, human behavior is complex and other environmental or social factors may influence food intake while drinking, he adds.
David Kareken and Robert Considine, neuroscientists at Indiana University, point out that although several human studies have shown drinking alcohol before a meal increases food intake, the reasons have remained unclear. They add these “elegant animal experiments” involved manipulations that could not be done in humans, and were needed to better understand how alcohol affects hunger. “This work sheds a lot of light on how that pre-meal aperitif might push us toward gaining a few extra pounds,” they wrote in an e-mail.
Building on this work, Cains is interested in why alcohol seems to make us crave certain foods, such as those greasy nachos. “I've never had a drink and then really fancied a salad,” she says, “It would be interesting to see if [intoxicated] mice have a preference for a high-fat diet.”

domain: medical


text:
A Slice Of Soho
While roaming the Soho area of New York we came on this scene. The buildings with their fire escapes looked like it could have been a hundred years ago except that the new Trade Center Building is going up at the end of the street.
October 19th, 2012
Viewed 19 Times - Last Visitor from Kista, 26 - Sweden on 02/09/2015 at 6:05 AM

domain: geography


text:
|Hampton and His Red Shirts
by Alfred B. Williams
Originally Published in 1935
Reprint Edition, 2015
paperback; 382 pages; illustrated
The "carpetbagger" government that ruled South Carolina from 1868 until it was overthrown in 1876 caused more destruction than the four years of the War Between the States. Judging by the record which these corrupt politicians left, continuance of their rule would have resulted in the irretrievable annihilation of the fruits of two centuries of labor, ingenuity, and courage. This book is a fascinating chronicle of how the people of South Carolina, led by former Confederate General Wade Hampton and his famous Redshirts, rose up to free themselves from the intolerable and dangerous conditions of the Reconstruction period.

domain: library


text:
Info on New Irish Horror ‘An Ceann Deireanach’ (The Last One)
‘An Ceann Deireanach’ (The Last One) takes place during The Great Famine, at it’s height in 1847. The story see’s our four main protagonists Tadhg, Seamus, Jim and Thomas flee their village in order to save their lives and their families after a revolt takes place on the turf field in which they work on for an absentee landlord, killing the estate manager. As the men journey through a hellish landscape they seek refuge inside a wood but soon through isolation and starvation their minds begin to break, but to what point?
“The Great Famine still remains a chapter of great importance and significance in our Nation’s history. A time of religious oppression and foreign rule, which scarred our landscape and our mentality. Professor Oonagh Walsh recently conducted a study on the links of the great famine and it’s connections to mental health. In a census report carried out in 1841 there were 1,600 inmates in district asylums after the famine by 1900 there were 17,000. The hell in which these people lived truly would have devastated their mental stability seeing loved ones starve to death, people being separated from their families to be put in workhouse’s. We want to explore that with our protagonists on this journey of escape and survival’ says Eamonn Tutty Co Producer, Writer & Director ‘An Ceann Deireanach’ (The Last One) of Reckoner Productions.
The film will be made in black and white with the use of natural lighting to capture the reality and bleakness of the time. The film will be recorded in Irish, something that will be a challenge for us as filmmakers, having to relearn a lot of what we learned in school. “By filming in Irish we hope to create a much needed revival of the Irish language on screen. As part of our cultural heritage, the language adds to the realism and poeticism the film is hoping to achieve”, says Dunne.” Drawing on influences like Andrei Tarkovsky’s “Mirror” and “Stalker” along with contemporary films like Steve McQueen’s “Hunger” and Ben Wheatley’s “A Field in England” we hope to create a film that will show the physical and mental deterioration that humans goes through when faced with such incredible hardship and sorrow. Although set during the 1840’s, the themes of hunger, survival, famine and mental illness are still relevant and universal in today’s world”.
Still in pre-production on their debut feature film, Eamonn & Alan have assembled a transmedia team to help raise awareness of their online funding campaign and to build and engage with an audience from prep to post production. They will update their journey by way of production diaries, cast & crew interviews, articles of relevance, production stills, and a timeline video each week documenting the history of the 1800’s up towards 1847 the height of the famine, and much more additional content.
Their funding campaign will end towards the end of March were they hope to raise €20,000 to begin production on ‘An Ceann Deireanach’ (The Last One) and it can be found here:

domain: artistic


text:
Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.
24 Related JoVE Articles!
Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure
Institutions: Children's Memorial Research Center, Northwestern University.
Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2
. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8
. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.
Medicine, Issue 61, Zebrafish, fetal alcohol exposure, Danio rerio, development, mRNA expression, morpholino, ethanol exposure
Measuring the Kinetics of mRNA Transcription in Single Living Cells
Institutions: Bar-Ilan University.
The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo
is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3
In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4
Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6
Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo
, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8
The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo
, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9
To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5
In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.
Cell Biology, Issue 54, mRNA transcription, nucleus, live-cell imaging, cellular dynamics, FRAP
Implantation of Total Artificial Heart in Congenital Heart Disease
Institutions: Texas Children's Hospital, Baylor College of Medicine, The University of Cincinnati College of Medicine.
In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital heart disease (CHD), the malformed heart presents a challenge to TAH implantation.
In the case presented here, a 17 year-old patient with congenital transposition of the great arteries (CCTGA) experienced progressively worsening HF due to his congenital condition. He was hospitalized multiple times and received an implantable cardioverter defibrillator (ICD). However, his condition soon deteriorated to end-stage HF with multisystem organ failure.
Due to the patient's grave clinical condition and the presence of complex cardiac lesions, the decision was made to proceed with a TAH. The abnormal arrangement of the patient's ventricles and great arteries required modifications to the TAH during implantation.
With the TAH in place, the patient was able to return home and regain strength and physical well-being while awaiting a donor heart. He was successfully bridged to heart transplantation 5 months after receiving the device. This report highlights the TAH is feasible even in patients with structurally abnormal hearts, with technical modification.
Medicine, Issue 89, total artificial heart, transposition of the great arteries, congenital heart disease, aortic insufficiency, ventricular outflow tract obstruction, conduit obstruction, heart failure
Protein Isolation from the Developing Embryonic Mouse Heart Valve Region
Institutions: University of North Carolina at Chapel Hill, New York-Presbyterian Hospital/Weill-Cornell Medical Center.
Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 μg of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.
Developmental Biology, Issue 91, heart, valve, embryonic, mouse, development, protein, western blot
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
Institutions: Max von Pettenkofer Institute, University of Cambridge, Ludwig-Maximilians-University Munich.
The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e.
total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila
, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.
Genetics, Issue 78, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis
Institutions: University of Nevada, Reno, St. Joseph's Medical Center, University of Rochester Medical Center .
Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e.
, medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroC
n of ischeM
sive to phaR
atment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.
Medicine, Issue 70, Anatomy, Physiology, Cardiology, Myocardial Ischemia, Cardiovascular Diseases, Health Occupations, Health Care, transient myocardial ischemia, Acute Coronary Syndrome, electrocardiogram, ST-segment monitoring, Holter monitoring, research methodology
Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Institutions: University of Florence, University of Florence.
Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.
Medicine, Issue 86, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure
Institutions: Catholic University of Leuven.
Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g.,
myocardial infarction), by pressure overload (e.g.,
systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.
Medicine, Issue 94, Myocardial infarction, cardiac remodelling, infarct expansion, heart failure, cardiac function, invasive hemodynamic measurements
Transthoracic Echocardiography in Mice
Institutions: Baylor College of Medicine (BCM), Baylor College of Medicine (BCM).
In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3
and as a result, in vivo
techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5
In addition, PWD is used to quantify localized velocity of turbulent flow,6
whereas TDI is used to measure the velocity of myocardial motion.7
Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.
Medicine, Issue 39, Echocardiography, pulse wave Doppler, tissue Doppler imaging, ultrasound
Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR
Institutions: UCSF/VA Medical Center, LoneStar Heart, Inc..
Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+
-Alginate and Ca2+
-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.
Medicine, Issue 74, Biomedical Engineering, Anatomy, Physiology, Biophysics, Molecular Biology, Surgery, Cardiology, Cardiovascular Diseases, bioinjection, ventricular wall stress, mathematical model, heart failure, cardiac function, myocardium, left ventricle, LV, MRI, imaging, clinical techniques
Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography
Institutions: Harvard Medical School, Chang Gung Memorial Hospital.
Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3
. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4
. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.
Medicine, Issue 84, Trans-thoracic echocardiography (TTE), right ventricle (RV), pulmonary artery constriction (PAC), peak velocity, right ventricular systolic pressure (RVSP)
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Institutions: Joint Unit Hospices de Lyon-bioMérieux, BioMérieux, Hospices Civils de Lyon, Lyon 1 University, BioMérieux, Hospices Civils de Lyon, Hospices Civils de Lyon.
The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1
. ‘How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2
or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4
. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g.
PCA3 in prostate cancer5,6
and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10
. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1
Medicine, Issue 81, Cancer Biology, Genetics, Molecular Biology, Prostate, Retroviridae, Biomarkers, Pharmacological, Tumor Markers, Biological, Prostatectomy, Microarray Analysis, Gene Expression, Diagnosis, Human Endogenous Retroviruses, HERV, microarray, Transcriptome, prostate cancer, Affymetrix
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis
Institutions: The University of Texas Graduate School of Biomedical Sciences at Houston.
Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro
. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro
and in vivo
Genetics, Issue 93, EML Cells, Self-renewal, Differentiation, Hematopoietic precursor cell, RNA-Sequencing, Data analysis
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Institutions: McGill University, Karolinska Institutet, McGill University.
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
Biochemistry, Issue 87, Cells, Eukaryota, Nutritional and Metabolic Diseases, Neoplasms, Metabolic Phenomena, Cell Physiological Phenomena, mRNA translation, ribosomes,
protein synthesis, genome-wide analysis, translatome, mTOR, eIF4E, 4E-BP1
Echocardiographic Assessment of the Right Heart in Mice
Institutions: Vanderbilt University Medical Center, Vanderbilt University Medical Center.
Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.
Medicine, Issue 81, Anatomy, Physiology, Biomedical Engineering, Cardiology, Cardiac Imaging Techniques, Echocardiography, Echocardiography, Doppler, Cardiovascular Physiological Processes, Cardiovascular System, Cardiovascular Diseases, Echocardiography, right ventricle, right ventricular function, pulmonary hypertension, Pulmonary Arterial Hypertension, transgenic models, hemodynamics, animal model
Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis
Institutions: Purdue University.
embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila
tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila
larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila
embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.
Biochemistry, Issue 85, Gene Expression, nuclei isolation, Drosophila, KASH, GFP, cell-type specific
Microinjection of Zebrafish Embryos to Analyze Gene Function
Institutions: Harvard Medical School, Children’s Hospital Boston.
One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo. Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product. Conversely, processed mRNA can be added to the embryo to increase levels of a gene product. The vehicle for adding either mRNA or morpholino to an embryo is microinjection. Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour. This video shows all the steps involved in microinjection. Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish. Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume. Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.
Developmental Biology, Issue 25, zebrafish, morpholino, development, microinjection, heart of glass, heg
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing

domain: health


text:
I read a post on TheBloggess.com that kind of wracked my soul for the day. Coming Out is about mental illness. My sister died 2 years ago from cancer. Before that she struggled with mental illness throughout her entire life and as a child had a myriad of different health problems to deal with including a heart transplant at the age of 8.
This is Heather:
She wrote a book about her life before she died. I'm reading through the draft this week as my mom is getting ready to actually publish it.
Recommended for You
Coming out about mental illness is a big deal. It's an inconceivable trial for those who suffer with it. For the families of those who have mental illness it's a journey about understanding, forgiveness, support and endurance.
People with mental illness cannot pray it away. They can't just "work harder" or "change their attitude" and make it all go away. It's a monumental exercise to manage it. Medications pile up on top of medications and make it a "crazy" mess to put it lightly.
So if you, those you love or those you are acquainted with struggle with mental illness. Come out. Help people understand. Help people have compassion for those who suffer. I've seen a lot of suffering and I stand in awe and amazement at what my sister endured and there are millions like her suffering who could really benefit from people taking some time to become enlightened about mental illness and what it really means.

domain: psychology


Home Topics Abdullahi Dikko
The Chief of Army Staff, Lt. Gen. Tukur Buratai, on Friday reiterated the commitment of the Nigerian Army to end insurgency in North-East and other security challenges in other parts of the country.
The trial of Balarabe Abdullahi and Usman Yusuf (currently at large) who are being prosecuted by the Economic and Financial Crimes, EFCC, for cloning the phone number of Abdulmumini Bako, a former special assistant to the former Comptroller General of the Nigeria Customs Service, Abdullahi Dikko, continued on March 5 with the prosecution presenting its third witness.
The high-profile individuals who are reportedly being probed by the Economic and Financial Crimes Commission (EFCC) include former governors, ex-ministers, justices and an ex-first lady.
Names of some high profile individuals being investigated by the Economic and Financial Crimes Commission, EFCC, have been revealed.
National Leader of the All Progressives Congress, (APC) Sen. Bola Tinubu, on Sunday said the election of Dr Kayode Fayemi in Saturday’s gubernatorial poll was a victory for the people.
Femi Kuti has spent his life railing in song against the ills holding back Africa. But these days he is feeling some optimism.
Oyo State Governor, Senator Abiola Ajimobi, has described the victory of Dr. Kayode Fayemi in the just-concluded gubernatorial election in Ekiti State as a demonstration of the people’s trust in the All Progressives Congress (APC) as a party that has the capacity to liberate the state from mis-governance and gross under-development.
The Edo State Chapter of the All Progressives Congress (APC), held a victory rally to celebrate the success of the party’s candidate in Saturday’s governorship election in Ekiti State, Dr. John Kayode Fayemi, who has been announced winner of the polls.
Socio-Economic Rights and Accountability Project (SERAP) has urged the Minister of Finance, Mrs Kemi Adeosun, to without further delay show responsibility and clear the air over the damaging certificate forgery allegations against her and settle the facts of the case once and for all.
The National Broadcasting Commission (NBC) has shut down the Ekiti Broadcasting Service, BSES.
The Minister of Federal Capital Territory (FCT), Alhaji Muhammad Bello, has reinstated Dr. Rilwanu Mohammed as Executive Secretary of Primary Health Care Development Board.
Mr Yekini Nabena, the Deputy National Publicity Secretary of the All Progressives Congress, has accused Senate President Bukola Saraki of playing the role of double agent in the APC.
President Muhammadu Buhari on Sunday in a congratulatory message to the winner of the Ekiti State governorship election, Dr. Kayode Fayemi, said Ekiti people had spoken loud and clear.
The management of Yankari Game Reserve, Bauchi has confirmed the death of two persons killed by elephants, near Bajama village close to the reserve.


——By Shedrack Onitsha————-
The immediate past Governor of Delta State, Dr Emmanuel Uduaghan has stated that against rumours going around, no one is leaving PDP in the state, adding that where their leader, Chief James Ibori tells them to go, is where they will go.
Uduaghan stated this weekend in Warri during the stakeholders’ meeting of the PDP Delta South Senatorial District at his home, warned party faithfuls against rumour mongering saying the party will not patronize blackmailers.
He noted that the party is stronger in the state despite the challenges at the national level while assuring that all the leaders of the party are united and that no one is leaving the party.
According to Uduaghan; “we will not patronize blackmailers, nobody is leaving PDP, where Ibori our leader says that we should go, that is where shall go.
He commended Governor Ifeanyi Okowa for the laudable projects he has executed despite taking over government at a difficult time.
He also Senator James Manger on his leadership role that ensured that the Maritime University got the federal government backing through the needed legal framework for its take off, noting that where the university is cited is immaterial because it will serve the entire Delta State and Nigeria.
The Chairman of the party in Delta South, Chief Emman Agbaduba appreciated party leaders and stakeholders for their support for the party, and urged them not to relent in their commitment to the party ahead of 2019.
The meeting was attended by party leaders and top government functionaries including, National Vice Chairman, South South, Elder Emmanuel Ogidi, State Chairman of the party, Barr. Kingsley Esiso, Senator James Manager, Minority Leader of the House of Representatives, Rt Hon Leo Ogor, Hon Daniel Reyenieju, members of the Delta State House of Assembly and, Council Chairmen.
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text:
Narcissus ‘Blushing Lady’ is a late season bloomer in the Jonquil section of Narcissus with multiple flowers per stem and a delightful fragrance. The flowers open light yellow and over time the cups gradually age to a light pink blush. This species will thrive in most of the soils found in the Chicago area in full sun or under the canopy of deciduous trees. In addition to Chicago winters, it is also very tolerant of the hot humid summers of the Deep South that cause many daffodils down there to rot. Free of most insect and diseases, rabbits and deer avoid it because of chemicals within the plant sap. Other groups of chemicals within the sap can cause contact dermatitis in sensitive individuals. Great for planting near a sidewalk, door or window where the fragrance can be appreciated.
March - April, May - June
Bedding or Border, Groundcover, Understory
Attracts Butterflies, Resistant To Deer, Native to Midwest

domain: biology


The UK saxophonist’s highly anticipated debut arrives with an eclectic and joyously grooving set of material.
Blue Note unearths an archival session from the Jazz Messengers, and it cooks, as expected.
The legendary spiritual jazz collective returns with a more somber, yet still groove-heavy set of music.
The Norwegian progressive jazz fusion group deliver a warmly exciting permutation of their consistent, yet constantly changing sound.
Expanded reissue includes 28 bonus tracks.
A gentle and meditative improvisational response to the Australian wildfires from earlier this year.
New album due out in August.
The London musician and producer downplays his ability on the keys on this nonetheless solid set of groove.
10 more additions to our list of fusion’s historical greats.
Hear new single “Pace.”
A meeting of four brilliant minds that feels too short for what it could be.
Hear a new interpretation of “Angels and Demons at Play.”
Plus a new documentary is on the way, documenting the making of ‘Universal Beings’.
The jazz legend has died at 78.
A powerful set of jazz rock that carries the heaviness of Sabbath with the innovation of Scandinavian jazz.
Hear new single “Freeze Tag.”
Hear their new version of 1979’s “Seductive Fantasy.”


text:
Welcome to our weekly forecast of weekend fantasy basketball! Every Friday, check here for some insight into the best FanDuel plays for the weekend. There's lot of games tonight so it's a great chance to play.
FanDuel is a great way to play fantasy basketball. It's new everyday, and so much fun! You get to pick your own 9-player lineup and get the chance to win some money!
Editor's Note: SB Nation's partner FanDuel is running a FREE money fantasy basketball league tonight . Half of the league win cash. Join now!
Click here to get started playing FanDuel
So, we hope you have a great Christmas and play FanDuel as well! Top heavy plays, medium plays and lowkey plays right here for you! Remember, just my take, do what you like!
HIGH - James Harden vs the Cavaliers
Cleveland is on a back-to-back and Harden will have to be super active and productive to keep the Rockets in this one. Look for him to score 30+ points, and see what he does with assists and rebounds.
MID - Brook Lopez vs the Blazers
Portland typically gives up a lot of fantasy points to center and Lopez has been playing good. He is usually a solid pick, but here I think he will exceed his value.
LOW - Tyler Johnson vs the Nuggets
Without Goran Dragic, the Heat will play Johnson more, hopefully. Udrih may still start, but it is probably time for him to get some extra burn and he could score well for them in this one.
HIGH - John Wall vs the Celtics
Boston doesn't really have a stopper at point guard. Isaiah Thomas should just get out of the way. Wall plays well on back-to-backs to make up for his team and should drop a good game here I think.
MID - Gordon Hayward vs the Lakers
At home, I see Hayward having a hay-day against a team that doesn't defend well. He should contribute all around and score well.
LOW - Jae Crowder vs the Wizards
On the flip side, I think Crowder will take advantage of how the Wizards will play a little small and lanky. Crowder can get easy bucket and pick up miscellaneous stats for you.
HIGH - Russell Westbrook vs the Heat
No one will be able to stop him. I don't want to talk about it.
MID - Karl-Anthony Towns vs the Suns
An up and down game will produce plenty of rebounds and block opportunities for Towns. He should even score well against a small front line.
LOW - Gerald Green vs the Thunder
The Heat will probably be playing from behind a lot, and so more bench time and more shots from Green. You don't get penalize for missing, so Green will take them.
I hope that helps you pick your team! Choose wisely, have fun and join us on FanDuel!
Remember, all this is my opinions and not that of Hot Hot Hoops, SB Nation or FanDuel. FD has given me some cash to start playing.

domain: sports


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The latest TODAY Book Club pick is "Positive," a memoir by Paige Rawl, who was diagnosed as HIV-positive when she was in middle school.
At 15, while battling depression in the wake of the bullying she faced, Rawl attempted suicide by taking one pill for each year of her life.
Fortunately she survived, and at 19 she is now an advocate and mentor helping to educate kids about the dangers of bullying and spreading awareness of HIV and AIDS.
"Positive" tells how Page Rawl battled bullying and fought her way back from her lowest low. The new memoir, published by Harper Collins Publishers, hit bookstores on August 26.
Read along with TODAY viewers, sharing your reaction to the book on Twitter via @TODAYsBooks and the TODAY Book Club Google+ community. Be sure to follow the TODAY Book Club newsletter for the latest information.
And we want to know how you stay strong and positive in your life. Send us a picture or a message with the hashtag #PositiveProject.

domain: news


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HBO Nordic updated a behind-the-scenes video yesterday which was related to the locations featured in Game of Thrones season 7. People in the US can’t watch this video, but Spanish broadcasting Moviestar had an international version up soon enough.
— Series en Movistar+ (@MovistarSeries) June 8, 2017
Now, Movistar has brought up something new for the fans from season 7, establishing itself as the media outlet that keeps on giving. In the Image below, Jaime and Cersei are seen standing near the giant map of Westeros we saw in the season 7 trailer. Cersei, we note, has some wine with her. She now may be a royal pariah to a dark queen, but some things always remain same.
Now, here is a picture of Daenerys Targaryen, taking a moment to touch the sand of her homeland after she lands at Dragonstone.
Another new image from the trailer. Also, Dany’s new black-and-red conqueror gown just refuses to quit.
— dragon's daughter (@yeahclarke) June 9, 2017
Finally, we have our man, with the real snow surrounding him.

domain: entertainment


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Emerging shares fall Apple on the background of General growth of the technology sector suggests that traders are pessimistic about the future of the company.
According to EN.insider.pro, 15 may, short positions in the shares of Apple rose $1.3 billion while during the same time period exchange traded Fund that tracks the overall technology sector, registered a decline in the number of short positions at $316 million
The last two days were especially successful for those who play against Apple as a result of falling stock by 6.2%, the amount of short positions has reached more than $500 million.
Given that Apple is superior to the other companies of technological sector, it is not surprising that she became the target of speculators,”bears.” By the middle of last week, shares of the iPhone maker rose by 34% YTD, compared with 8.7% in the case of S&P 500.
From that moment on 9 January 2007 Steve jobs presented the world the first iPhone price of Apple stock increased more than 12 times, from $12 to $145. the iPhone forever changed the world and the company itself. Since that time the company’s shares and its market capitalization is valued highly.
Of course, when the iPhone was a familiar device, investors have come to expect from Apple’s revolutionary device, which also could change the world. Unfortunately, presented to the public in late April 2015, the Apple Watch, this device did not. This device did not cause changes in the minds of customers and disappointed investors, who began a massive sale of Apple shares.
It was not until 27 July 2016, when Apple celebrated the sale of their one billionth iPhone and stock prices have moved to growth, having overcome a breakaway. Later in 2016 the fiasco with the release of the Samsung Galaxy Note 7 has given impetus to growth of Apple shares, which was fully apparent during the craze of the shares of the U.S. stock market after the election of the President of the United States, in the Wake of the frenzied rise in prices due to the bright prospects of the presidency of the trump. At this point, Apple has returned to its former glory, when its shares rose to a peak of $135, recovered to the level preceding the presentation of the disappointing Apple Watch.
The current sale of shares of technology companies has forced investors to think that it may be a prelude to the collapse of the market after Friday, Goldman Sachs released a report in which its analysts found similarities between the current market situation and conditions prevailing before the collapse of the dot-com
As of 21:30 GMT, Apple shares traded at $145,43 (-0,79%).

domain: finance


In the ever-evolving landscape of digital marketing and SEO, the importance of content cannot be overstated. Among the various types of content strategies that have emerged, long-form content has emerged as a significant player, reshaping the way websites are perceived by search engines and online audiences alike. In this article, we’ll delve into the role of Long-Form Content in modern SEO and uncover why it has become a key strategy for achieving higher search rankings, driving organic traffic, and engaging users.
Understanding Long-Form Content
Long-form content refers to articles, blog posts, guides, and other pieces of content that go beyond the traditional short-form approach. While short-form content is often concise and quick to consume, long-form content is characterized by its depth, detail, and comprehensive coverage of a topic. It typically exceeds 1,500 words and can extend to several thousand words, offering an in-depth exploration of the subject matter.
The SEO Benefits of Long-Form Content
1. Comprehensive Coverage and Authority
Long-form content allows you to provide a comprehensive and authoritative treatment of a topic. By addressing various aspects, nuances, and angles of a subject, you showcase your expertise and demonstrate a deep understanding of the subject matter. This can elevate your website’s credibility and authority in the eyes of both search engines and readers.
2. Keyword Opportunities and Search Intent
Long-form content provides ample space for integrating relevant keywords naturally. This is crucial for improving your website’s visibility in search engine results pages (SERPs). Additionally, as search engines increasingly focus on understanding user intent, long-form content enables you to address multiple facets of a search query, enhancing the chances of your content matching various user intentions.
3. Dwell Time and User Engagement
Long-form content has the potential to keep users on your page for a longer duration, known as dwell time. When readers spend more time engaging with your content, search engines interpret it as a positive signal, indicating that your content is valuable and relevant. This can contribute to improved search rankings.
4. Backlink Magnet
High-quality long-form content often becomes a magnet for backlinks. Other websites and bloggers are more likely to link to well-researched, informative, and authoritative content. Backlinks are a crucial factor in SEO, as they indicate to search engines that your content is worth referencing.
5. Reduced Bounce Rates
Long-form content tends to have lower bounce rates compared to short-form content. When users find comprehensive and valuable information on your page, they are more likely to explore different sections of your content, reducing the likelihood of bouncing back to the search results.
Crafting Effective Long-Form Content
Creating successful long-form content requires a strategic approach:
1. Thorough Research
In-depth research is the foundation of long-form content. Gather reliable sources, data, and insights to support your claims and provide accurate information.
2. Clear Structure
Organize your content with clear headings, subheadings, and bullet points. This enhances readability and helps users navigate through your content effortlessly.
3. Engaging Introduction
Grab the reader’s attention from the outset. A compelling introduction sets the tone for the rest of the article and encourages readers to continue reading.
4. Visual Elements
Incorporate visuals such as images, infographics, and videos to break up the text and make your content visually appealing.
5. Internal and External LinksInclude both internal links to other relevant pages on your website and external links to authoritative sources. This enhances the user experience and provides additional value.
6. Conclusion and Call to Action
Summarize key points and provide a clear call to action at the end of your content. This could be a request to subscribe, share, or explore related content.
As search engines become more sophisticated and user expectations evolve, the role of long-form content in modern SEO continues to grow. By offering depth, relevance, and value, long-form content not only satisfies search algorithms but also engages and educates your audience. Embracing long-form content as a core component of your SEO strategy can lead to improved search rankings, higher organic traffic, and a more engaged readership.


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Chipola College, Tallahassee Community College, and nonmetropolitan industry leaders join with the Florida State University Information Use Management and Policy Institute and School of Library and Information Studies to examine the educational and career pathways of information technology (IT) technicians supporting broadband deployment in communities in Northwest Florida. The mixed methods research project investigates the region's broadband staffing needs to identify the roles of broadband technicians, education needed to develop the knowledge and skills to be successful in these roles, and the process to sustain long-lasting partnerships between educational and industry stakeholders. The pervasive nature of broadband indicates a need for IT/broadband workers who can handle a broad range of issues associated with the needs of commerce and communities in the region. In addition, the study conceptualizes and establishes the use of the term, nonmetropolitan, to describe communities that are adjacent to metro or urban areas and comprise a population of both rural and urban residents. The study deliverables include 1) a gap analysis that supports curriculum development; 2) creation of a network of nonmetropolitan stakeholders that includes employers, industries, community institutions, and technician educators; 3) an iterative curriculum/labor needs assessment that ensures depth and breadth of findings; 4) multiple faculty/industry workshops that deliver recommendations and solicit faculty/industry review of research findings; and 5) an array of activities that supports broad dissemination of the findings to inform local educators and industry, and engage all stakeholders in ongoing discussion. The research study contributes to a growing, though limited body of knowledge on the intersecting needs of IT technicians, educators, and employers in broadband technologies.
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BREAKING: Protesters attack Google bus in West Oakland, smashing window
J Thoendell stashed this in Tech
Tensions between Bay Area activists and tech institutions ratcheted up this morning when protestors attacked a Google bus in West Oakland, smashing the bus’s rear window while Google employees were inside. Local news organization KQED reports that Google has confirmed the attack.
It does seem like there is growing tension:
As the tech industry is growing in size, wealth, and power, it attracts more people to the SF Bay Area and decreases the amount of available housing. Some San Francisco and East Bay residents are getting pushed out of the cities they live in because they can no longer afford rent. Likewise, some landlords are taking advantage of a law called the Ellis Act to force long time residents out and sell the property for high rates.
Many employees who work for Google, Apple, Facebook, LinkedIn, and other tech companies based on the Peninsula live in San Francisco or the East Bay. They take shuttles run by the corporations from these locations down to work in Menlo Park, Palo Alto, and Mountain View.
As a result, the private shuttles have become a symbol of the socioeconomic conflict unfolding in the Bay Area. Protestors have blockaded tech company buses on previous instances before today, most notably a few weeks ago when a fake video went viralonline. The clip showed a supposed Google employee shouting back at protestors, “You can’t afford it? You can leave.” The man turned out to be a union organizer performing a bit of activist theater.
And it'll get worse, especially if the tech companies beef up on security. Goons with pepper spray and tasers... mix in plenty of activists willing to provoke an assault... that occupy Wall Street movement may start to spread.


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Rolls-Royce envisages developing a new smaller engine based on core demonstrator applications being run in Germany, but is vague on plans to commit the powerplant to potential re-engined offerings from Airbus and Boeing.
Speaking at the annual ISTAT aircraft appraisers in Orlando, R-R civil aerospace president Mark King said he is convinced the market should see a new small engine from the company after 2015.
King said the new engine might power a re-engined aircraft. But he hopes to see the new powerplant on a new airframe: "[You] have to look at the whole system front to back [to take advantage of new technology]," he said.
Further out, King said R-R believes open rotor technology is a potential game-changer. The company has completed three sets of wind testing of open rotor technology in conjunction with airframers and is convinced that noise issues associated with the engine are "definitely addressable", although King admitted that challenges remain in integrating the engine to an aircraft.
King declined to commit on a specific time period for the introduction of open rotor technology until aircraft manufacturers decide on a configuration for the engine. At that point, said King, R-R could explain "what our open rotor configuration would look like".


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Waves quietly breaking on a forgotten beach in the West fjords. Drops rising above the horizon. The story of Asterias. Black terrain. Rhythmical waves.
Everlasting cycle of the water that has no beginning and no end.
The latest Asterias Collection was inspired by waves quietly breaking on a forgotten beach in the West fjords resulting in the most stunning range of simultaneously light, feminine and intricate necklaces with modern, three dimensional designs evoking natural patterns. With the choice of a gold silver plate, silver oxidized and silver finish in several designs the Asterias range compliments differing tastes and suits several styles. Choose from a kyanite, onix or ruby to personalise your Asterias necklace, while increasing the importance of the piece to you.
We will notify you on events like Low stock, Restock, Price drop or general reminders so that you don’t miss the deal

domain: design


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St. Seraphim of Sarov, arguably, is the most popular of Russian saints. July 19 marks the celebration of the first uncovering of the relics of St. Seraphim. This event took place in the year 1903 in Imperial Russia. That day in Imperial Russia, 70 years after his repose, or death, the physical presence of his relics brought about miraculous healings among those present.
The primary gospel reading of this Sunday came from St. Matthew’s Gospel (Mt 9: 27 – 35). In this reading we hear of the healing of two blind men Read the rest of this entry »

domain: history