God Bless Texas. What else can you say about the second greatest state in the Union. This is the state that gave us the Alamo and Ted Cruz. This is the state that if the Obama Regime were not interfering, could single handedly lead the nation out of the Great Obama Depression.
Good things come out of Texas. Now something really good has come from Texas.
What is it?
Last night, a Federal Judge in Texas issued a restraining order blocking Barack Obama’s executive Amnesty. Judge Andrew Hansen wrote that the Plaintiffs (the states with real Americans who sued to stop Obama) would suffer irreparable injury if the law were allowed to go forward before the courts had adjudicated the matter.
Score one for the good guys. Former Texas Attorney General and now Texas Governor Greg Abbot led the fight. Abbot is one of the sane Republicans who understands what Amnesty will do to America.
The order on the injunction will immediately be appealed to the 5th Circuit Court of Appeals in New Orleans. Fortunately among the active judges of the 5th Circuit, there is a 2 to 1 majority of judges appointed by Republicans. There is a strong probability that the injunction will be upheld by the 5th Circuit and probably the Supreme Court too, though that will only be by a 5-4 vote.
The real question now is what will the Regime do?
The law has never been a barrier to the actions of Barack Obama. He has deliberately ignored numerous laws and simply rewritten laws if he saw fit. Will Obama just ignore the ruling of the Court in Texas?
Given his narcissism, there is no telling.
If the Republicans in Congress had any sense, they would now pass the Homeland Security funding bill with one addition. They should attach a rider prohibiting the government from spending any money to appeal Judge Hansen’s decision or to even defend the lawsuit.
Meanwhile, God bless Texas!


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People find yourself needing extra funds at some point in our everyday lives. While many individuals don’t such as the notion of borrowing, other people understand exactly exactly how helpful taking out fully that loan can especially be whenever you’re hit with unanticipated expenses. Utilizing the appropriate information, the probability of getting a much better deal are greater, therefore make certain you’re well informed before making a decision to simply take a loan out.
Installment loans are usually considered one of the more helpful loans you can easily go for, and they’re one thing you should think about if you’re reasoning about a house renovation, spending money on a marriage, or obtaining a brand new vehicle.
FAST AND SIMPLE UNSECURED LOANS AS MUCH AS $2500
An installment loan is one of the most popular forms of loans, that requires borrowing a specific amount of cash, and repaying it with interest over a number of fixed payments called “installments. ”
These re payments are often made month-to-month, nevertheless they may be made regular, bi-weekly, or for a various routine that is determined prior to taking out of the loan.
Installment loans are mostly utilized to pay for mid-to-high costs such as for online installment wy instance purchasing a house that is new automobile. These prices are frequently a great deal to manage simultaneously, so getting an installment loan and spreading re re re payments away over a extensive duration can cause them to become more manageable.
The bucks you will need at ninja speed.
Common Kinds of Installment Loans
Installment loans are actually a few of the most common kinds of loans available:
Auto loans are accustomed to buy a vehicle that is new. Monthly payments are often compensated over 12 to 96 months, but not all loan providers problem loans within that range. In the event that you neglect to take care of the payment schedule, your newly bought automobile will act as collateral, as well as the loan provider may repossess it.
Mortgages are installment loans useful for purchasing a house. They’re usually paid back over 15 to three decades with monthly premiums. Just as in auto loans, your home will act as security if you can’t result in the payments that are monthly. Mortgages frequently have reduced interest levels because they are considered guaranteed, and some have even fixed prices that don’t modification as time passes.
Unsecured loans can be utilized for a number of purposes, like debt consolidating or settling sudden unforeseen costs like medical bills. They don’t require collateral like your automobile or household, nevertheless they include slightly greater interest levels.
EASY AND QUICK UNSECURED LOANS AS MUCH AS $2500
Just How Do Installment Loans Work?
Lenders require some type or sort of insurance that you’ll have the ability to pay off the lent quantity, and that’s why some loans need security. Most lenders also require which you have working work or other type of earnings, to make sure that you’ll be in a position to spend each installment.
With respect to the loan provider, you may make re re payments through automatic month-to-month deductions from your banking account, direct deposit during the shop, or higher the device.
Paying down each installment on time can help you enhance your credit history, which could be useful the next time you’re taking out fully that loan. A greater credit rating ensures that you are able to borrow a greater quantity or negotiate a much better interest.
The bucks you will need at ninja speed.
Things to Think About When Getting an Installment Loan
Getting mortgage which allows you to definitely make affordable payments that are monthly be a concern whenever taking out fully an installment loan. Loan providers think about numerous facets to determine their danger and provide you with an interest that is specific according to your credit history, earnings, as well as other debts. The main concern for loan providers is ensuring it is possible to repay the mortgage.
Rates of interest and terms when it comes to loan will be different according to your credit score as well as the certain loan provider. Short-term loans typically give higher rates of interest than long-lasting loans. However if it is possible to spend from the loan early you can easily avoid extra interest.
Which is why it is essential to assess your financial predicament and figure out exactly what is best suited for you personally. Many people desire to repay the mortgage at the earliest opportunity, although some choose to extend it down for smaller re payments as time passes. Whichever you decide on, make certain it fits your allowance, and it has a payment plan you’re confident with.
Remember that the lent quantity is fixed, even if you have the ability to refinance your loan to receive extra funds and a brand new rate of interest. CreditNinja does not have penalties for repaying your loan early, though some other loan providers might ask you for extra costs. CreditNinja additionally typically permits installment borrowers to refinance.

domain: economics


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Industry response to the Ukraine Crisis
Posted: 29th March 2022
This note has been developed as the first of a series by the Construction Leadership Council to support the response of the UK construction industry to the impacts of the Ukraine crisis. The CLC is monitoring the situation carefully and, working with government, will determine appropriate responses as developments occur. The CLC’s Product Availability Group (PAG) has increased the frequency of its meetings to fortnightly in anticipation of rapid changes in materials markets.
PROBLEMS AND CONSEQUENCES
At time of publication (week commencing 28th March 2022), the timescale and extent of disruption associated with the invasion are unknown, and the potential for knock-on effects affecting industry capacity, logistics networks and client demand. What is becoming clear is that significant Ukrainian industrial capacity has been destroyed during the conflict. This means that there will be some long-term market impacts, such as reduced availability of steel in the mainland European market.
The situation is evolving very quickly and is being influenced by an even wider range of factors. The recent fall in energy prices for example has been influenced by an anticipated short-term slowdown in China triggered by Covid-19 lockdowns. It is recommended that all businesses regularly monitor key market indicators including energy and commodity prices to keep informed of developments.
Materials: In the UK, it is anticipated that the withdrawal of materials and products sourced directly from Russia, Ukraine and Belorussia will have a limited impact on markets. The total share of construction material imports is less than 2%. However, there are some critical categories – the most important of which are bitumen, cast iron products, rebar and timber.
Energy: Wholesale energy prices in the UK have been directly affected by supply constraints to Europe and other issues associated with the operation of the European energy market. These are having a direct impact on the costs of manufacture of energy intensive products including steel, cement, bricks and blocks and glass. Energy markets are also being disrupted by sanctions affecting energy trading intermediaries.
Indirect impacts resulting from disrupted raw material and energy supply chains can already be felt and are expected to have a more serious impact. Some raw materials produced in Russia, including copper and timber, are processed in Europe and enter the UK supply chain via firms within the EU
- Disruption to established supply chains: Locating raw materials from a tight market that has lost capacity as a result of sanctions/buyer preference limiting sourcing from the conflict zone (e.g., iron ore and pulverised coal, timber).
- Loss of access to fixed price agreements and hedges: Disruption to pricing models and short-term requirements to secure raw materials at spot market prices.
- Reduced production volumes: Manufacturing shutdowns due uneconomic operation due to high energy costs.
The potential implications of both direct and indirect impacts on the UK construction sector are significant. Construction enterprises at all scales, from the largest tier one contractor to the smallest R&M specialist are being affected. The materials supply and distribution segment is also at risk. The main challenges are:
Rapidly increasing costs affecting the viability of projects or pain/gain share on target cost projects.
Unexpected cost increases affecting bids and projects on-site, which could result in losses if fixed price protection is not in place.
Inflexible or imbalanced commercial terms, such as very short, fixed price periods preventing suppliers, sub-contractors and main contractors from obtaining fixed price protections in bids and change orders.
Potential for survivalist behaviours and perhaps even business failure as a result of product availability issues, increased costs and other commercial pressures. Any of these outcomes could result in less obvious safety and quality compromises that are often associated with projects that are facing financial difficulties.
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Telephone: 020 7399 7400
- Clarification on Professional Construction Consultants demonstrating competence
- Government must make up for lost time with an improved plan for healthier, climate resilient homes and buildings
- CLC sets out ambitious delivery plan for 2024
- Three new appointments to Supply Chair Sustainability School Board
- Education and training in the UK plumbing and heating industry


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TheCute Abiola also known as Lawyer Kunle has debut a new web comedy series titled HUSTLE 247. This episode is titled DRIVER JOB SECURED.
KABI and Okele finally secured a driving job, it is safe to say they are just so unlucky at this point. which job can they finally do to make a good living ? let us keep up with this wonderful series, new episode drops next Sunday.
CLICK HERE TO COMMENT

domain: entertainment


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While there is no general agreement upon which is the greatest film of all time,
many ... Many films are widely considered among the best ever made, whether
they appear at #1 on each list or not. Fo...
Feb 28, 2011 ... TOP 100 BEST MOVIES EVER MADE .... crusader must come to terms with one
of the greatest psychological tests of his ability to fight injustice.
Ebert's Best Film Lists1967 - present. ... are my personal favourite films. They are
in no way th greatest films ever made -. 1) Raging Bull (1980) - Martin Scorsese.
Jun 25, 2014 ... Hollywood insiders were asked to pick their favorite movies of all time, providing
us with a ranked list of 100 of the best movies ever made.
The Best of RT tool finds the top reviewed films of all time in any ...
Filmsite.org's Tim Dirks has spotlighted the 100 all-time greatest movies, many ...
Possibly the most influential thriller ever made, Alfred Hitchcockâ€™s tale of a ...
Tomatometer rankings of the top 100 best movies of 2016 and all time. Lists of
recent ... We list the Top 100 movies ever based on their Tomatometer Score.
The very first edition of AFI's 100 Years...100 Movies is a list of the 100 greatest
American films of all time.
The best movies of all time, ranked by movie experts and film fans alike. What are
the greatest movies of all time? This list of the top films ever made was created ...
Jul 20, 2015 ... BBC Culture polled film critics from around the world to determine the best
American movies ever made. The results are surprising – Gone With ...


MetriCide® 28 Glutaraldehyde High Level Disinfectant (Gallon)
- PLEASE NOTE, orders are shipped first come, first served.
- Reusable for up to 28 days
- Recommended for immersible and heat-sensitive equipment and instruments
- Contains a surfactant
- No dilution required
- Activation Required
MetriCide 28 is intended for use in a tray system with a variety of noncritical, semi-critical, and critical devices – including anesthesia equipment, respiratory therapy equipment, metallic equipment or instruments, rubber objects, plastic objects, and thermometers.
MetriCide 28 may be reused up to 28 days, assuming the minimum effective concentration of glutaraldehyde, as measured by a chemical indicator, remains within acceptable parameters and other conditions of use are met.
MetriTest™ 1.8% Test Strips are a glutaraldehyde concentration monitor for use in glutaraldehyde-containing germicide solutions with a minimum effective concentration (MEC) of 1.8% glutaraldehyde. It is recommended to test the glutaraldehyde solution prior to each use to help prevent healthcare-acquired infections (HAIs).
Brand: Metrex | MPN: 10-2800
How many? One gallon


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Actor Chris Pratt dished out some life advice to fans as he collected the Generation Award at the MTV Movie and TV Awards.
The Jurassic World: Fallen Kingdom star picked up the accolade previously given to fellow Hollywood A-listers including Will Smith and Robert Downey Jr at a ceremony in Los Angeles.
During his acceptance speech, Pratt thanked his family before listing out his nine most essential life tips.
He began: "Number one: Breathe. If you don't, you will suffocate. Number two: You have a soul. Be careful with it."
"Number three: Don't be a turd. If you are strong, be a protector. If you are smart, be a humble influencer. Strength and intelligence can be weapons, so do not wield them against the weak. That makes you a bully. Be bigger than than that.
"Number four: When giving a dog medicine, put the medics in a little piece of hamburger and they won't even know they're eating medicine.
"Number five: It doesn't matter what it is. Earn it. A good deed. Reach out to someone in pain. Be of service. It feels good and it's good for your soul."
"Number six: God is real. God loves you, God wants the best for you. Believe that; I do."
For his seventh piece of advice, Pratt told the audience how to go to the toilet at a party.
He then added: "Number eight: Learn to pray. It's easy, and it is so good for your soul."
The 38-year-old actor concluded: "Nobody is perfect. People will tell you that you are perfect just the way that you are, you are not! You are imperfect. You always will be, but there is a powerful force that designed you that way, and if you are willing to accept that, you will have grace.
"And grace is a gift, and like the freedom that we enjoy in this country that grace was paid for with somebody else's blood. Do not forget that. Don't take that for granted."
Black Panther was the big winner at the awards ceremony, winning four prizes from seven nominations. It bagged the biggest prize of the night, Best Movie, as well as won Best Hero (Chadwick Boseman), Best Villain (Michael B Jordan) and Best Performance In A Movie (Boseman).
While accepting the Best Hero award, leading star Boseman handed over his prize to a man who saved lives when he fought off a gunman who opened fire in a Nashville restaurant. During his acceptance speech, the 40-year-old called James Shaw Jr on to the stage.
Shaw Jr disarmed an attacker armed with an assault rifle at a Waffle House in Antioch, Tennessee, in April. Four people were killed in the attack and suspected gunman Travis Reinking was later arrested away from the scene.
Handing over the award on stage in Los Angeles, Boseman told Shaw Jr: "This is going to live at your house."
He later tweeted: "During the show, I was honored to meet @JamesShawJr9. He saved countless lives by tackling a gunman who opened fire on people in Antioch, Tennessee in April. He's a true hero that walks among us."
Netflix's sci-fi drama Stranger Things picked up three gongs on the night, Best TV Show, Best Performance in A Show for its star Millie Bobby Brown and Most Frightened Performance for Noah Schnapp.
Here is the full list of the winners at the 2018 MTV Movie And TV Awards:
Scene Stealer - Madelaine Petsch (Riverdale)
Most Frightened Performance - Noah Schnapp (Stranger Things)
Best Hero - Chadwick Boseman (Black Panther)
Best Kiss - Nick Robinson and Keiynan Lonsdale (Love, Simon)
Best Comedic Performance - Tiffany Haddish (Girls Trip)
Best Reality Series/Franchise - Keeping Up With The Kardashians
Best Villain - Michael B Jordan (Black Panther)
Trailblazer Award - Lena Waithe
Best Onscreen Team - IT
Best Performance In A Show - Millie Bobby Brown (Stranger Things)
Generation Award - Chris Pratt
Best Performance In A Movie - Chadwick Boseman (Black Panther)
Best TV Show - Stranger Things
Best Music Documentary - Gaga: Five Foot Two
Best Movie - Black Panther


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Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.
27 Related JoVE Articles!
Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats
Institutions: Wright State University, Universidade São Judas Tadeu.
Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e.
, increased sensitivity to noxious stimulus, and allodynia i.e.,
hypersensitivity to normally innocuous stimuli1
. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2
. However, as stated by Bölcskei et al
, determination of "pain
" in animal models is challenging due to its subjective nature3
. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3
. For instance, by using the hot-plate method of Ankier4
, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 °C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5
. As the recently described method of Bölcskei et al.6
, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Almási et al 5
and Bölcskei et al 3
. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.
Neuroscience, Issue 63, Diabetes, painful diabetic neuropathy, nociception, thermal nociceptive threshold, nocifensive behavior
Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Institutions: University of Western Ontario.
The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1
. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3
. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4
. Habituation is stimulus specific5
. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6
. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7
. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10
. Both habituation and PPI are disrupted in patients suffering from schizophrenia11
, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13
. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15
Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16
, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.
Neuroscience, Issue 55, Startle responses, rat, mouse, sensory gating, sensory filtering, short-term habituation, long-term habituation, prepulse inhibition
High Throughput Microinjections of Sea Urchin Zygotes
Institutions: University of Delaware .
Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.
Developmental Biology, Issue 83, Sea Urchins, microinjection, sea urchin embryos, treatment delivery, high throughput, mouth pipette, DNA constructs, mRNAs, morpholino antisense oligonucleotides
A Method for Microinjection of Patiria minata Zygotes
Institutions: Carnegie Mellon University.
Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata
, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus
and Lytechinus variegatus
. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata
, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.
Developmental Biology, Issue 91, Embryology, Patiria miniata, sea star, echinoderm, development, gene regulatory networks, microinjection, gene expression perturbation, antisense oligonucleotide, reporter expression
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)
Institutions: University of New Hampshire, Massachusetts Division of Marine Fisheries, Boston University, Middle College.
Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female's seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George's Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.
Environmental Sciences, Issue 84, sperm limitation, spermatophore, lobster fishery, sex ratios, sperm receptacle, mating, American lobster, Homarus americanus
Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish
Institutions: University of Maine.
The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo
quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.
Immunology, Issue 79, Phagocytes, Immune System, Zebrafish, Reactive Oxygen Species, Immune System Processes, Host-Pathogen Interactions, Respiratory Burst, Immune System Phenomena, innate immunity, bacteria, virus, infection]
Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis
Institutions: Harvard University.
The amphipod Parhyale hawaiensis
is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale
has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster
. In contrast to the syncytial cleavages of Drosophila
, the early cleavages of Parhyale
are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale
embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale
embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale
cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.
Developmental Biology, Issue 85, Amphipod, experimental embryology, micromere, germ line, ablation, developmental potential, vasa
Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries
Institutions: University of Missouri, Dalton Cardiovascular Research Center.
The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.
endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+
signaling, which leads to production of diffusible autacoids (e.g.
nitric oxide and arachidonic acid metabolites)1-3
that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+
signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ
to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7
. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.
Basic Protocol, Issue 81, endothelial tubes, microcirculation, calcium signaling, resistance vasculature, Confocal microscopy
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Institutions: Uppsala University, Swedish University of Agricultural Sciences.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ
hybridization, a technique used to localize cell specific mRNA expression. The in situ
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies
). Here we present a modified DIG in situ
hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana
and Brassica napus
. The protocol worked equally well for the species and genes studied. AtAP3
were observed in second and third whorl floral organs in A. thaliana
and B. napus
and DAL13 in microsporophylls of male cones from P. abies
. For P. abies
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Plant Biology, Issue 26, RNA, expression analysis, Norway spruce, Arabidopsis, rapeseed, conifers
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards
Institutions: Macquarie University.
Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.
Neuroscience, Issue 2, Visual sensitivity, motion perception, operant conditioning, speed, coherence, Jacky dragon (Amphibolurus muricatus)
Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana
Institutions: Wageningen University, Huazhong Agricultural University.
Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens,
a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum
), its related wild tuber-bearing Solanum
) and the model plant Nicotiana benthamiana
. In addition to functional analysis of single genes, such as resistance (R
) or avirulence (Avr
) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R
transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium
, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium
. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.
Plant Biology, Issue 83, Genetics, Bioengineering, Plants, Genetically Modified, DNA, Plant Immunity, Plant Diseases, Genes, Genome, Plant Pathology, Effectoromics, Agroinfiltration, PVX agroinfection, potato, Nicotiana benthamiana, high-throughput, functional genomics
Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm
Institutions: Trier University, Trier University.
Selection tasks in which simple stimuli (e.g.
letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.
Behavior, Issue 87, stimulus-response binding, distractor-response binding, response retrieval, distractor inhibition, event file, action control, selection task
Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, USDA-ARS Dairy Forage Research Center, USDA-ARS Pasture Systems Watershed Management Research Unit.
Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.
Environmental Sciences, Issue 90, greenhouse gas, trace gas, gas flux, static chamber, soil, field, agriculture, climate
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Institutions: Colorado State University, Oak Ridge National Laboratory, University of Colorado.
Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e.
C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e.
colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ
soil type and temperature can influence enzyme kinetics.
Environmental Sciences, Issue 81, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
Training Synesthetic Letter-color Associations by Reading in Color
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
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
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals
Institutions: University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign.
An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis
and M. faveolata
. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae
endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis
and M. faveolata
contain similar types of chlorophyll and chromatophores. However, M. annularis
and M. faveolata
exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.
Environmental Sciences, Issue 91, Serial block face imaging, two-photon fluorescence microscopy, Montastraea annularis, Montastraea faveolata, 3D coral tissue morphology and structure, zooxanthellae, chromatophore, autofluorescence, light harvesting optimization, environmental change
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
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
Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Institutions: Yale University, Virginia Tech, The Hebrew University of Jerusalem.
The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2
. Plant litter comprises the majority of detritus3
, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5
. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6
. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9
because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6
. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7
. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10
, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6
. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira
), a dominant grasshopper herbivore (Melanoplus femurrubrum
),and a variety of grass and forb plants9
Environmental Sciences, Issue 73, Microbiology, Plant Biology, Entomology, Organisms, Investigative Techniques, Biological Phenomena, Chemical Phenomena, Metabolic Phenomena, Microbiological Phenomena, Earth Resources and Remote Sensing, Life Sciences (General), Litter Decomposition, Ecological Stoichiometry, Physiological Stress and Ecosystem Function, Predation Risk, Soil Respiration, Carbon Sequestration, Soil Science, respiration, spider, grasshoper, model system
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
Institutions: University of Houston, Michigan State University.
The scaling of body parts is a central feature of animal morphology1-7
. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bxα, where x and y are the size of the two traits, such as organ and body size8,9
. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + αlog(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of α, called the 'allometric coefficient'9,10
. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and α) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12
Not all traits scale linearly with each other or with body size (e.g., 13,14
) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila
, our methodology should be applicable to nearly any fully metamorphic insect.
Developmental Biology, Issue 56, Drosophila, allometry, morphology, body size, scaling, insect
A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)
Institutions: Michigan State University, U.S. Geological Survey.
Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus
) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.
Developmental Biology, Issue 88, Biliary atresia, liver development, bile duct degeneration, Petromyzon marinus, metamorphosis, apoptosis
Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies
Institutions: University of California, Irvine (UCI).
Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (>80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.
Immunology, issue 8, blood, cell isolation, natural killer, lymphocyte, primary cells, negative selection, PBMC, Ficoll gradient, cell separation

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domain: design


By Frank Wiener
New platforms enable service providers to continue to leverage TDM services using established methods, while migrating to the new generation of Ethernet services in a phased, controlled, and more cost-efficient manner.
Service providers globally have a common goal of a network that delivers an expanding array of packet-based services in a capital and operationally efficient manner. The challenge for most providers is the amount of TDM-based infrastructure remaining in their network and how to transition this infrastructure to achieve that goal. Up until now carriers have lacked a compelling option to exploit this installed infrastructure and associated human capital while scaling and optimizing the network for packet transmission.
Many providers have taken an overlay strategy where all new network scale is addressed with a parallel overlay packet network. Unfortunately, this approach actually increases the costs and headaches for service providers as it results in two networks to plan, maintain, and operate. Others have taken a protocol convergence approach, translating TDM services into packet transports with pseudowire in a quest to evolve to an all-packet switching and transport infrastructure. However, the cost and complexity of translating TDM services to packet for transport and then back to TDM for handoff at the other end of the network adds cost and complexity—with no additional revenue.
|FIGURE 1. Multilayer management provides a converged view of the network or per-layer and interlayer perspectives.|
Until recently, there were limited alternatives to these two approaches. Fortunately, new packet optical networking platforms (PONPs) provide the ability to natively support any mix of both TDM and packet capabilities in one homogeneous network. This enables service providers to continue to leverage TDM services using established methods, while migrating to the new generation of Ethernet services in a phased, controlled, and more cost-efficient manner.
Multilayer PONPs enabling new options
New PONPs are modular and leverage the latest component technologies to integrate scalable, next-generation packet transport switch functions and DWDM/ROADMs with SONET/SDH multiservice provisioning platforms (MSPPs). Any mix of packet, TDM, and DWDM transport modules can be deployed on an as-needed basis. Packet modules support both wholesale and retail Ethernet transport services. MSPP modules support native SONET and SDH transport functions. DWDM modules support expansion of capacity for all service types. ITU G.709 Optical Transport Network (OTN) technology provides a common digital wrapper for both packet and TDM services and is ideal for transport over DWDM fiber links.
New innovations in packet optical transport technologies support multilayer management to enable a unified management view of the multiple technology and operational layers that exist in virtually every large network (see Fig. 1). These systems visually display the layers and relationships between conduits, fibers, wavelengths, OTN, SONET/SDH, connection-oriented Ethernet transport, and service layers of the network to enable management optimization across all layers of the network.
The ability to natively groom packet, TDM, and optical services in one integrated platform and then transport these services over a common fiber-based network enables the efficiency and operational benefits of one fully converged network. These systems operate seamlessly alongside the installed base of SONET or SDH equipment. Transparency ensures that both TDM and packet services are transported predictably and reliably. The payload data is simply encapsulated and transported. There is no TDM-to-packet processing-related jitter or latency. The costly, complex, and risky circuit emulation techniques required to transport high-bandwidth SONET/SDH payloads over packet are unnecessary and can be avoided entirely in the transport network.
This packet optical transport approach offloads transport traffic from routers, which are often used to provide Layer 2 switching and transport. Offloading transit traffic unclogs expensive router ports, freeing this capacity for network services expansion. Packet optical networking platforms can now provide lower latency with these services for a fraction of the cost of using routers for transport. The platforms also provide higher capacity, lower latency and jitter, and native support for established SONET/SDH services on a lower-cost network that scales better and is simpler and cheaper to operate.
Achieving network and operational convergence and making more efficient use of network resources, while TDM and packet technologies natively coexist in one physically converged network, sets the stage for a simplified and cost-optimized network transition from TDM to packet.
Multilayer transport network transformation strategies
Modular PONPs integrate into existing networks to provide incremental service capacity and facilitate network transformation to a packet-services-optimized network. Multiple strategies coexist during the transition—as determined by the existing and emerging network and business constraints that vary across the network.
While sequencing can vary, the basic elements of the network transformation strategy will likely consist of the following benefits for service providers:
- deploying Metro Ethernet Forum (MEF) services us ing connection-oriented Ethernet (COE) transport
- consolidating and further scaling SONET with OC- 192 or SDH with STM-64
- providing gateway functions to bridge services be- tween TDM and packet transports
- using OTN as the common high-bandwidth transparent transport technology for its forward error correction; consistent robust operations, administration, and maintenance (OAM); and its high-capacity optical services channel for manage ment connectivity
- expanding fiber capacity with DWDM
- leveraging next-generation node architectures to scale services and transport capacity
- optimizing and simplifying with multilayer management
Scale MEF services with COE transport for efficiency
Ethernet has become the de facto interface for both retail and wholesale packet transport services. Multiservice provisioning platforms introduced multiservice transport by supporting encapsulation of packet services for grooming using SONET. Grooming is a term associated with transport networks and supports aggregation of traffic over shared high-capacity links. Grooming of packet services using SONET/SDH is complex and suboptimal. Next-generation PONPs support grooming of conventional and advanced packet services such as Ethernet Private Line, Ethernet Virtual Private Line, and Ethernet LAN over a native packet transport technology.
|FIGURE 2. Packet optical networking platforms support MEF services with COE transport|
Transport grooming supports the sharing of capacity, while providing total independence of the different customer payloads, with strong service-level agreement (SLA) guarantees. As a result, transport switches have distinct and critical requirements not found in enterprise or even carrier-class service switches. Connection-oriented Ethernet transport combines packet grooming capability with the transport attributes of SONET and SDH, and is enabled by Ethernet standards such as 802.1Qay (PBB-TE) and the evolving transport-focused MPLS variants such as T-MPLS and MPLS-TP (see Fig. 2).
Grooming of multiple lightly loaded Gigabit Ethernet (GbE) and 10GbE services onto aggregated transports improves efficiency and reduces cost. Packet transport OAM, combined with OTN overhead, actually improves the quality of the product by improving optical performance, reducing jitter and latency, and supporting superior visibility and control of the network for increased availability and reduced time-to-repair.
Consolidate SONET with OC-192 (SDH with STM-64)
While COE is expected to rapidly rise as the preferred Ethernet transport standard, installed infrastructure, existing contracts, and customer agreements will continue to drive demand for SONET/SDH-based transport services. In addition, legacy subrate services often consume valuable fiber resources. Many service providers consider the option of upgrading legacy MSPPs to OC-192/STM-64 or introducing new MSPPs. However, these upgrades are often costly and further proliferate network complexity with multiple separate systems with limited long-term Ethernet potential.
New PONPs use the latest integrated circuit designs that provide the functionality of historic MSPPs on a blade—with disruptive low cost. These integrated SONET/SDH MSPP modules set the stage for a seamless migration to packet by sharing a common management infrastructure and the ability to map Ethernet-over-SONET/SDH to transport Ethernet services over existing SONET/SDH infrastructure.
|FIGURE 3. Packet optical networking platforms provide the gateway between packet and TDM.|
Bridge MEF services across COE and SONET with gateways
Packet optical networking platforms now provide the ability to transport MEF services, SONET/SDH, OTN, and COE services. Because multiservice and multitechnology support are not sufficient, PONPs must also support concurrent nonblocking grooming of these different services and technologies.
These platforms should also provide multiplexing and adaptation functions to support interlayer functions. For example, gateway network elements will be required between parts of the network that use Ethernet-over-SONET transport and other parts that support COE (see Fig. 3). The gateway nodes may be required to groom in both the TDM and packet domains at the same time. This functionality provides new levels of flexibility to provision services across multiple transport technologies and to seamlessly facilitate the eventual transition of services from one transport technology to another.
Expand fiber capacity with DWDM
Packet optical networking platforms provide several options to multiplex optical channels over shared fibers. Fixed and tunable transceivers, pluggable and nonpluggable optics, and fixed and tunable multiplexing and demultiplexing methods together support both colored and colorless DWDM grooming and both directional and nondirectional transceiver deployments. No single combination of these technologies can target all of the applications. And the technology will continue to evolve.
Through innovative design, new developments in PONPs support four or eight 10-Gbps optical channel DWDM configurations to provide simple and cost-effective capacity expansions. As services scale, modular upgrades to 40-channel configurations with wavelength-selectable switching enable scale with unrivaled operational simplicity.
Optimize and simplify with multilayer management
As we explore the transformation from TDM to packet and even post-transition packet networks, it becomes clear that networks will consist of multiple layers. At a minimum, these networks will consist of:
- service layers
- transport multiplex layers
- OTN layers
- optical wavelength layers
- fibers and conduits
Historically, these separate layers have been planned and managed independently, often by different dedicated resources. As PONPs provide the ability to interface with networks across these layers, innovative multilayer management systems provide the traditional fault, configuration, accounting, performance, and security (FCAPS) functions for these layers to simplify operations.
Integrating these layers in multitechnology node hardware has significant economic advantages. These advantages are even more dramatic when the node and network software are also multilayer aware. Software can also provide the ability to model the network, cluster demands, and analyze resource assignments across each layer and between the layers of the network. Grooming and multiplexing optimize the economics of all services and all layers of the network.
Network operators are tasked with configuring and maintaining these networks. What-you-see-is-what-you-get, three-dimensional visual-display technology helps network operators understand the network environment and the new technologies. Clarity reduces error, increases confidence, and empowers people to do their jobs.
The transformation to come
The network transformation from TDM to packet will happen at different rates across different parts of the network. Innovations with PONPs present new opportunities to accelerate directly to advanced packet services using packet transport. These systems may also provide the gateway functions to bridge Ethernet services across a mix of TDM and COE transports to facilitate a more evolutionary approach to network transformation.
As network capacities continue to scale, support for an easy expansion to DWDM provides a scalable approach that can extend across the network. Finally, integrated multilayer management systems round out the process with the ability to manage and provision services across both TDM and packet transport with one integrated operational view of the network, enabling a flexible and more holistic approach to network transformation.
Frank Wiener is the vice president of marketing and business development for Cyan. He has a diverse background ranging from engineering to product management, marketing, sales, business development, general management, and executive management for Nortel, AT&T/Paradyne, Calix, and Cyan. Wiener also has authored three telecom books.
Links to more information
LIGHTWAVE:Packet Optical Transport Systems: The new POTS
LIGHTWAVE:What Features Should Packet Optical Transport Equipment Contain?
LIGHTWAVE ONLINE: Infonetics: Packet Optical Transport and 40G Represent Big Opportunities for Non-Incumbent Vendors


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Saipem (OTCMKTS: SAPMF – Get a rating) has been updated by Zacks Investment Research from a “sell” rating to a “hold” rating in a research report released Tuesday to clients and investors, Zacks.com reports.
According to Zacks, “Saipem SpA provides drilling services as well as engineering, procurement, construction and installation of pipelines and complex projects, onshore and offshore, primarily in the oil and gas market. Saipem SpA is headquartered in Milan, Italy.”
Shares of Saipem stock traded down $0.05 during Tuesday trading hours, hitting $1.24. 525 shares of the company were traded, against an average volume of 5,331. Saipem has a 52-week low of $1.07 and a 52-week high of $2.85. The company has a fifty-day moving average of $1.22 and a 200-day moving average of $1.70.
About Saipem (Get a rating)
Saipem SpA provides energy and infrastructure solutions worldwide. The company operates through five divisions: Offshore Engineering & Construction (E&C), Onshore Engineering & Construction, Offshore Drilling, Onshore Drilling and XSIGHT. It offers engineering, construction, installation of platforms, pipelines, subsea fields, maintenance, modification, operation and dismantling, as well as the development of offshore wind farms and energy integration projects.
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domain: finance