text:
Can I Have My Saline Breast Implants Replaced With Silicone Implants Through An Axillary Incision?
Q: Dr. Eppley, I would like to know your experience with breast augmentation revisions. Is this something that you have done often? I am considering revision from saline to silicone and believe I’d like to go smaller. My previous surgical site for the augmentation was axillary and can this be done again with a revision? Any information you can provide would be greatly appreciated.
A:Any plastic surgeon that performs a substantial number of breast augmentations will perform secondary breast implant surgery, given desires of changes in size later in life, change in breast shape due to pregnancy and weight loss and the eventual failure of most breast implants over a patient’s lifetime. So yes I have done many breast implant exchanges and revisional surgery.
I believe you are asking if your existing saline implants could be replaced with smaller silicone implants through your already present axillary scars. The answer to that question is maybe…and it depends on what size silicone implant needs to be inserted through such a small incision. In days past, I would say it would not be adviseable unless the silicone implants are fairly small (under 350ccs). But with today’s funnel insertion technique, much bigger silicone breasts implants can now be placed through much smaller incisions.
Dr. Barry Eppley


text:
The flood situation remained grim in certain parts of north Karnataka following torrential rains.
Many rivers, rivulets and streams were in spate on Friday as water was released from the Tungabhadra Dam, sources said.
According to sources, water-level rose in Hirehalla rivulet, trapping five farmers on an island in Koluru village. The farmers had gone to remove the pump-set when suddenly the water-level increased and they got marooned. Soon, the others informed the police, which sent a team from the fire and emergency services.
The team rescued the five and brought them to the safety.
In Vijayapura district, a bridge was washed away and some villagers went there after learning that it had developed cracks and may not withstand the flow in the Sogli rivulet.
The bridge was connecting Sogli with Mookihal in Taalikote Taluk.
Reports said onions grown in a large swathe of land in the Bagalkote district were washed away due to floods.
In Yadgir, farmers got emotional before the Central team which went to inspect and prepare a report on the rain-related damage. The farmers showed to the team the damaged crops.

domain: geography


One of the Anonymous hacker, who was exposed after providing evidence for the Steubenville, Ohio Rape Case- is facing more time in jail than the sexual offenders.
A Video was leaked by Deric Lostutter (hacktivist), which was made by the rapists themselves, and clearly showing their 16-year-old victim was unconscious and unable to consent.
FBI raided the home of that 26-year-old “hacktivist” last April and he admitted about the distribution of Video.
As Deric leaked the video which helped catch the convicts in the rape case, for that he is facing 10 Years in jail which also included obtaining tweets and posts on social media contained the information regarding the rape case convicts.
Above we have learned that Deric is facing 10 Years Jail for the Video Sharing, but now, you will be shocked after hearing -one of the rape convict named ‘Ma’Lik Richmond’ was released from the Jail for ‘Good Behavior,’ he was released from less than a year.


text:
A new report commissioned by the Swedish Environmental Protection Agency says Sweden needs at least 300 wolves to maintain a healthy population.
But it also adds that new blood is needed for the pack.
Maria Hörnell Willebrand heads up the wildlife analysis department at the agency. She says the report was launched at the request of the government, which wanted to know what the minimum necessary population was to ensure the long-term health of the species and what the socio-economic consequences of having that population would be.
To find that out, the agency had two different teams of scientists – one from the United States and the other from Sweden – carry out their own investigations.
Finland, Russian bloodlines needed
According to the agency’s latest population census, there are some 400 wolves living in the center of the country. But they are in need of new bloodlines from other wolf packs found to the east in Finland and Russia.
“Today (the Swedish wolf population) looks OK. But we need to be sure that we have at least one immigrant every five years to have this low number of wolves and still have a favorable reference population,” Willebrand tells Radio Sweden.
Currently, there is one male and female pair that wondered into Sweden from Finland during the winter of 2012.
Willebrand adds that she doesn’t see Tuesday’s report affecting the right for local county boards to set quotas for wolf hunts.
Related stories from around the North:
Canada: Fighting to protect bird habitat in North America’s boreal forest (SLIDESHOW), Eye on the Arctic
Finland: New measures to protect the Saimaa seal in Finland, Yle News
Iceland: Endangered whale meat shipped from Iceland via Halifax, The Canadian Press
Norway: Rapid growth in Svalbard walrus population, Barents Observer
Sweden: Wolves attack sheep and lambs in North Sweden, Radio Sweden
United States: Banned pollutants turn up in Alaska fur seals, Alaska Dispatch News

domain: biology


text:
Keto Diet Shopping List: What Foods to Buy and Avoid
The ketogenic diet (keto diet) is a low-carb, high-fat diet that has gained popularity in recent years. It involves drastically reducing carbohydrate intake and replacing it with healthy fats. This shift in macronutrient balance triggers a metabolic process called ketosis, where the body starts burning fat for fuel instead of glucose.
Following a keto diet can offer several benefits, including weight loss, improved blood sugar control, reduced inflammation, and enhanced cognitive function. However, it's important to approach this diet with a well-rounded understanding of the foods to include and avoid.
Keto-Friendly Foods: What To Buy
A. Protein Sources:
Meat: Opt for unprocessed and grass-fed options like beef, pork, and lamb.
Poultry: Choose chicken, turkey, or duck without added sugars or breading.
Fish and Seafood: Include salmon, tuna, mackerel, sardines, and other fatty fish.
Eggs: Select organic and free-range eggs for optimal nutrient content.
B. Healthy Fats:
Olive Oil: Use extra virgin and cold-pressed olive oil for cooking and dressings.
Avocado and Avocado Oil: Incorporate avocados and their oil into your diet.
Nuts and Seeds: Enjoy almonds, walnuts, chia seeds, and flaxseeds as snacks or additions to meals.
Coconut Oil and Butter: Use coconut oil for cooking and grass-fed butter for flavor and healthy fats.
C. Low-Carb Vegetables:
Leafy Greens: Include kale, spinach, lettuce, and arugula in your salads and meals.
Cruciferous Vegetables: Broccoli, cauliflower, Brussels sprouts, and cabbage are excellent choices.
Non-Starchy Vegetables: Zucchini, cucumber, celery, and bell peppers are low in carbs and high in nutrients.
Mushrooms and Tomatoes: Add mushrooms and tomatoes to your dishes for flavor and texture.
Blueberries, Raspberries, Blackberries, and Strawberries: These berries are low in carbs and rich in antioxidants.
Foods To Avoid On The Keto Diet
A. Sugary Foods:
Processed Foods: Avoid cookies, cakes, candy, sugary cereals, and other processed items.
Sweetened Beverages: Steer clear of soda, juice, energy drinks, and sweetened teas.
Artificial Sweeteners: Limit or avoid artificial sweeteners like aspartame, sucralose, and saccharin.
B. High-Carb Foods:
Grains: Eliminate bread, pasta, rice, quinoa, and oats from your diet.
Starchy Vegetables: Avoid potatoes, corn, peas, and other high-carb vegetables.
Legumes: Beans, lentils, and chickpeas are high in carbs and should be avoided.
Fruits: Limit high-sugar fruits like bananas, grapes, and mangoes.
C. Unhealthy Fats:
Trans Fats: Found in processed foods and fried foods, trans fats are harmful to health.
Vegetable Oils: Soybean oil, corn oil, and canola oil are high in omega-6 fatty acids and should be avoided.
Margarine and Shortening: These processed fats are unhealthy and should be replaced with natural fats.
Tips For Keto Diet Shopping
Read Food Labels Carefully: Check for hidden sugars and carbs in packaged foods.
Shop the Perimeter of the Grocery Store: Fresh produce, meat, and dairy are often found there.
Buy in Bulk: Save money and reduce packaging waste by purchasing in bulk.
Plan Your Meals Ahead of Time: This will help you avoid impulse purchases and ensure you have the right ingredients on hand.
Experiment with New Foods: Try different vegetables, nuts, and seeds to find new favorites that fit your keto lifestyle.
Adopting a keto diet requires careful planning and a focus on consuming nutrient-rich foods while avoiding processed and high-carb items. By following the guidelines provided in this article, you can create a well-rounded keto diet that supports your health goals. Remember to consult with a healthcare professional before starting a keto diet, especially if you have any underlying health conditions.

domain: health


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This is Dusty Duke playing bluesy, dirty/southern rock seasoned with a hint of country. Not bad mixture at all. My copy arrived last week and it has been under heavy rotation since. Troublemaker is Dusty Duke’s second release and that debut album is now on my “want list”. As far as I know, this is limited to 150 copies and still available via their BandCamp site and from Black Farm Records (debut & this second album available via both sources). […]
Love at first sight.. but I have to admit that it took way too long to purchase this bluesy/stoner release from Shotgun Sawyer. Never been even near States but somehow this record manages to take me there.. weird, but must have something to do that bluesy sound. Really happy that I finally decided to grab this record. This is still available from the source, Ripple Music (US) and regular black version available from their EU shop (if you want to […]
A damn nice blues/hard rock album from this australian band. I listened this album via BandCamp few times and result of those moments can be seen on the picture above. Bluesy rock with touch of darkness, what else can I ask? Highly recommended album even though this has nothing to do with stoner/doom/sludge. Jus give a listen and you know what I mean. Still available from Kozmik Artifactz on white vinyl if you are interested. Yeah!!
Yeah, what a way to start this year. This came on last Friday and just look how awesome it looks! Pure STB quality when it comes how vinyl albums should be released. Been listening this a lot during today.. Damn good sounding heavy/hard/blues/stoner album and I already know that this album will have numerous spins in this household. This die-hard edition is limited to 105 copies and came with bonus twelve-inch, “A Flagrant Disregard For Happiness”. This package wasn’t cheap […]
These two albums came last week from Magnetic Eye Records. Usually I do not buy compilations, tribute albums etc. but with these two I just had to make an exception. I like both of these albums and records like these are quite easy way to check out new bands (yes, there are bands I have never hear about before). I know that there have been discussions on some forums about delay of this [Redux] version.. Hey, we all knew the […]
Even though I have said that I’m not that interested of test pressing.. here’s one, surprise. Very strong performance from this band on their second full-length released by Riding Easy Records. If you like good old-time rock favored with blues I suggest you to check this one out. These test pressings are limited to 30 copies (still wondering why so much..) and surprisingly sold out but there are still clear copies (limited to 200 copies) available from Riding Easy. Nice […]
This arrived today. Couldn’t resist purchasing this one after listening only one track, “Journey”. Damn good debut album from this Danish band. Bluesy rock’n’roll, awesome sounding vocals and those tunes played with cursed organ.. Yes, I want more! Released by Kozmik Artifactz and limited to three different colorways; black, orange and marbled (like this one I got). All variations still available from Kozmik — —.
Yes, yes, yes.. another test pressing from Riding Easy Records.. Few days ago I find myself thinking why in hell anyone want to buy test pressing and surprisingly this one arrived.. Slow Season and their debut album, very strong performance when talking about blues/stoner rock. I had this one on my wantlist for a while and then, suddenly this TP came available to purchase.. and here it is. I like this one, I like every damn record Riding Easy is […]
Yeah.. this came today. I honestly think that STB Records has taken this whole “Die Hard” concept to the next level lately (and others follow if they can.. or care). Quality release with outstanding packaging (came with extra mp3 coupons etc.). Thank You Steve, for making my day (once again)! Usually I’m not that big fan of split releases but with this album I’m very pleased. Stoner, blues rock, psychedelic (or whatever these two bands are performing) tunes hits you hard […]
And here’s a pretty damn good album filled with bluesy rock, seasoned with some psychedelic rock’n’roll. For a debut album this is quite good release, in my opinion and I have been listening this a lot lately. I really dig these retro sounding, bluesy, garage bands and The Highway Kind is turning to be one of my favorite. Album came in perfect condition & packaged secured which always makes me happy. According to their BandCamp page this is limited to […]
Look what I got from Riding Easy Records yesterday. Yep, the debut album from The Picturebooks and it sounds awesome. This German duo knows their Rock’n’Roll, bluesy kind. I really dig this album a lot, it sounds raw, it sounds dangerous, you can smell the sweat on it.. Thanks to Riding Easy for putting this jewel out, without You I would have missed this band totally. This clear version was limited to 50 copies and surprisingly it’s sold out but […]
And then I got this beauty from STB Records (with tons of extra stuff, mp3 coupons etc.). This is limited to 50 hand-numbered copies on clear vinyl with yellow & green splatter, and was available on Halloween’s eve. Actually I already have this on die hard version but I just couldn’t resist purchasing this.. damn me and my hoarding issues. Well, I love this album and really appreciate everything STB Records is doing. Nice item and really awesome record! —
It’s here! Countless mornings this record has been on my headphones on my way to work. More I listen to it, more I love it. Psychedelic-Blues-Rock played just like I love to hear it. Actually I received this album few weeks ago but there was some scratches on the vinyl so I decided to contact Steve from STB Records and today I received this replacement vinyl (really happy about this!). It’s awesome how DIY label like STB takes care of […]
Got this nice blues rock LP by Lonesome Shack today. Yeah, it’s limited & colored, but.. first corner.. second corner.. third corner.. Oh, carton stiffeners were used.. usually these are at least size of the cover.. not this time.. and of course we have one tiny seam split. This is reminder for me why I usually note the seller that I would like my purchases sent records & covers separated.. Finally, these words printed on the mailer, they do not […]
With this one I had a problem, I’m not that big fan of live recordings, but here it is. These purple, hand-numbered copies were available for few hours from Outer Battery records. And about the record, even though my despise towards live recordings, this actually sounds very nice. So, I’m quite happy that I managed to purchase this one. This is limited to 200 copies on red and 200 copies on purple vinyl. Both versions seems to be sold out. […]
And little different record (if I compare this to those I have been recently purchasing) this time. Sounds great, really like the singer’s voice (reminds me of Nick Cave and maybe a hint of Stuart Ashton Stables) and record is very enjoyable from start to end. This is something I have missed for some time now. Recommended, darken, bluesy rock! I do not know about total limitation of this release but 100 of those came on pink vinyl (sold out). […]
I went out to local record store for the latest album of Oranssi Pazuzu.. Well, they did not have that and on my way out of the store my eyes spotted this one.. Pristine copy and no excuse to leave this one there. Even though I haven’t been listening Danzig for years now, I had to buy this. Maybe I have to pay a visit to that store more often (last summer I found few copies of Mark Lanegan’s earlier […]
And here it is, the winner! Even though this ain’t the original copy (but 2010 unofficial release) I still damn love this album! Record full of awesome songs and so dark and hopeless. Original vinyl version is so damn expensive that I have to settle with this one. This is The Album. And there is, nor never will be song like “Black Wings”
This album has its place in my heart. It was the period of my life when I listened Jesus Lizard, Shellac and other so-called “noise” rock bands. I even love the cover of the album.. For me this is Blues Explosions coolest album. Get this, it’s still floating at the record store near you.
Found this band about three years ago. I was amazed by the fact that I didn’t knew anything about this act. But now I know much much much more. Records like this one keeps me trying to find more and more beauty from this darkness. Recommended, highly. As far as I know this color is from 2nd press and limited to 1000 copies. Available from straight from their website.
I remember reading something about this band somewhere.. can’t remember where or when. I picked this from the record store while I was visiting my grandmother up north. Nice pick without listening this before. I do not know anything about placing bands to certain genres but I would say that this is hazy blues rock mixed with little bit of psychedelia. You can get this from Boomkat, Norman Records, Insound and of course from Fonal Records (where you can find lots […]
I clearly remember when I first time heard these tunes. I was spending my time in library and in music department there was this record. I liked the cover so much that I borrowed the album. Year was 1994 and this record/artist totally stole my heart (well, more likely part of it)! Amazing album. Some might say that this was his turning point to worse but for me all his albums are brilliant!!
Well, this arrived today. Bought this from Discogs.. not as good condition as seller described (for me Near Mint or M- means that there is no visual marks on cover nor record..) but I can deal with it, fortunately it was quite inexpensive. Great record, been hunting this some time and now I got it, finally.

domain: entertainment


text:
Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.
20 Related JoVE Articles!
Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung
Institutions: University of Liverpool , University of Liverpool .
There is growing concern about the relevance of in vitro
antimicrobial susceptibility tests when applied to isolates of P. aeruginosa
from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1
. However, during chronic lung infections in CF, P. aeruginosa
populations exist in biofilms and there is evidence that the environment is largely microaerophilic2
. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3
Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa
growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6
. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa
based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
An ASM assay was developed in a microtitre plate format. P. aeruginosa
biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa
isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3
. Several in vitro
models have been used previously to study P. aeruginosa
. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9
. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2
and affect antibiotic susceptibility10
. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.
Immunology, Issue 64, Microbiology, Pseudomonas aeruginosa, antimicrobial susceptibility, artificial sputum media, lung infection, cystic fibrosis, diagnostics, plankton
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Institutions: University of Illinois Chicago - UIC.
Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa
, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa
are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8
. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11
. In this example, we use an RND protein, the P. aeruginosa
MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.
Cellular Biology, Issue 46, multidrug resistance, membrane protein, purification, transmembrane transport, MexB, detergent solubilization, protein detergent complex
Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection
Institutions: University College London.
The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5
. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7
. We developed a new model1
which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd
) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p
) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~
10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.
Immunology, Issue 80, Engineering, Technology, Diagnostic Techniques and Procedures, Early Diagnosis, Bacterial Infections and Mycoses, Lipids, Amino Acids, Peptides, and Proteins, Chemical Actions and Uses, Diagnosis, Therapeutics, Surface stress, vancomycin, mucopeptides, cantilever sensor
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)
Institutions: University of Ottawa.
This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro
. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture. In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.
Immunology, Issue 83, biofilm, planktonic, antibiotic resistance, static, antibacterial, minimal inhibitory concentration (MIC)
In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa
Institutions: CNRS & Université Paris Descartes.
There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa
in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.
Biochemistry, Issue 84, membrane protein, transport, antibiotic resistance, liposomes, proton gradient, bacteriorhodopsin
Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice
Institutions: San Raffaele Scientific Institute, Italian Cystic Fibrosis Research Foundation.
A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa
agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo
. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa
and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa
clinical strains in the agar-beads in vitro
, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa
RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa
colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.
Infection, Issue 85, Opportunistic Infections, Respiratory Tract Infections, Inflammation, Lung Diseases, Cystic Fibrosis, Pseudomonas aeruginosa
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis
Institutions: Marshall University.
is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa
is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa
is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa
. To better understand alginate regulation, we describe a protocol using the mini-himar1
transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1
transposon (pFAC) from host E. coli
SM10/λpir into recipient P. aeruginosa
biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas
isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE
(PA4033) and kinB
(PA5484), in strain PAO1 with a wild-type mucA
encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ22
). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.
Immunology, Issue 85, Pseudomonas aeruginosa, alginate, mucoidy, mutagenesis, mini-himar1 mariner transposon, pFAC
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
A Visual Assay to Monitor T6SS-mediated Bacterial Competition
Institutions: Imperial College London .
Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2
. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3
. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae
T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7
. Another striking example was recently documented using Pseudomonas aeruginosa
which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10
In the latter case,
three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1
). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10
. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa
, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14
, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli
DH5α) allowing a-complementation of the lacZ
gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.
Infection, Issue 73, Microbiology, Immunology, Infectious Diseases, Molecular Biology, Genetics, Biochemistry, Cellular Biology, Bacteriology, Bacteria, Type Six Secretion System, T6SS, Bacterial Competition, Killing Assay, Pseudomonas aeruginosa, E. coli, lacZ, CFU, bacterial screen, pathogens, assay
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Institutions: Delft University of Technology, Delft University of Technology.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli
and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9
addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli
to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2
) of the alkane hydroxylase system from Gordonia
were transformed into E. coli
. For the conversion of long-chain alkanes (C15-C36), theladA
gene from Geobacillus thermodenitrificans
was implemented. For the required further steps of the degradation process, ADH
and ALDH (
originating from G. thermodenitrificans
) were introduced10,11
. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli
K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii
OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g.
under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n
-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli
to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
Bioengineering, Issue 68, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract
Institutions: University of Texas Southwestern Medical Center , Harvard Medical School, University of Texas Southwestern Medical Center .
(PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1
. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2
, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo
transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3
in order to identify changes in bacterial gene expression during alterations to the host’s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4
. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.
Immunology, Issue 55, Pseudomonas, RNA, murine, cecum, transcriptome, qPCR, RT-PCR, PCR
One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures
Institutions: Maastricht University Medical Center, Erasmus Medical Center.
Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1
. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4
. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5
. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa
and Escherichia coli
as well as Gram-positive bacteria including Staphylococcus
spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp.,
and Streptococcus pneumoniae
could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus
spp. and (facultative) aerobe Gram-negative rods6
. This assay was based on a study in which PCR was used to measure the growth of bacteria7
. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1
). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.
Immunology, Issue 65, Infection, Medicine, Microbiology, Bacteria, real-time PCR, probes, pathogen detection, blood culture, 16S rDNA gene, antibiotic resistance, antibiotic susceptibility testing
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Microtiter Dish Biofilm Formation Assay
Institutions: Dartmouth Medical School.
Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae
, Escherichia coli
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa
. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.
Immunology, Issue 47, Biofilm, assay, bacteria, fungi, microtiter, static
Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells
Institutions: Danish Technical University, University of Copenhagen.
Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1
. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro
3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.
Immunology, Issue 47, Biofilm, Pseudomonas aeruginosa, Bacteria, Yeast, Saccharomyces cerevisiae, Flow cell system, Confocal Lases Scanning Microscopy, Microbiology, FLO11, Systems biology
Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells
Institutions: Dartmouth College, Indiana University Purdue University Indianapolis.
Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa
biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa
is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa
and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa
strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa
infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa
biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.
Cellular Biology, Issue 44, biofilm, Pseudomonas aeruginosa, airway, epithelial cells, co-culture, cytotoxicity, Cystic Fibrosis, virulence
Pseudomonas aeruginosa Induced Lung Injury Model
Institutions: University of Illinois at Chicago, Emory University, University of Illinois at Chicago.
In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa
or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa
injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.
Immunology, Issue 92, Lung, injury, pseudomonas, pneumonia, mouse model, alveoli

domain: medical


text:
DES MOINES, Iowa — “It seems appropriate on the fifteenth day of the fifth month to get out and celebrate Des Moines.”
To celebrate, the Bulldogs were out in the community, serving.
“I think it’s important for us to just remind folks that we appreciate all that Des Moines does for us for our community we certainly get a lot from them and aren’t afraid to give back.”
So Drake Athletic Director Brian Hardin and about 20 members of the athletic department took to the classrooms at East High.
“We’re going to be moving desks and other things around in their school so what normally would be done by three people will have more than 20 folks be able to do it.”
That includes football coach Todd Stepsis, who brought his two daughters with to help out, or ride the dolly.
They got to work quickly, splitting into groups of ten, sticking to social distancing as best as they can, and wearing masks.
“What would take them several days or weeks we’ll get done in a matter of hours today.”
This group wasn’t the only one from Drake giving back, they had a food drive in front of the Knapp Center, and members of the department also did their own services. Another sign that we can get through this pandemic together.
“I think it’s always important to give back to your community but now more than ever it’s critically important it reminds us how much good there is in this community. And this is a tough time but people of Des Moines and Drake Athletics are going to be tougher than what this virus is and we will come back through this better on the back end. Today is just a reminder of the great people and great things there are that we can do in our community and we’re excited to make a positive impact.”
515 day started a couple of years ago, and Hardin hopes it continues to grow.

domain: sociology


SQFlash 710 Full-size MiniPCIe PCIe/NVMe SSD
- PCIe/NVMe PCIe Gen3 x2 SSD
- Full-size MiniPCIe
- Compliant with PCIe Gen. III x2 interface and NVMe 1.2
- Support StrongECCTM (SECC) of ECC algorithm
- GPIO preserved for security function control
- GUI management tool & software API package
Advantech SQFlash 710 series Full-size MiniPCIe PCIe/NVMe SSD (Solid State Drive) delivers all the advantages of flash disk technology with PCIe Gen3 x2 interface, including being compliant with Full-size MiniPCIe form factor. The device offers a wide range of capacities up to 1TB and its performance can reach up to 1600 MB/s (for read) and 450 MB/s (for write) based on Toshiba 3D NAND (BiCS3) flash. The lower power consumption makes it an ideal storage choice for high performance demanding mobile devices.


NEW CASTLE, Del.—TA Instruments Inc. said its Discovery X3 Differential Scanning Calorimeter provides more data and consolidates three instruments into one.
The addition to the company's Discovery Differential Scanning Calorimeter incorporates its Fusion Cell and Tzero-brand technologies to handle up to three samples at the same time.
"The demand for the development of high-performance materials is increasing rapidly throughout many industries," the company said in a statement. "New materials require considerable testing and these tests significantly extend the development and validation timelines. Traditional DSCs are limited to analyzing a single sample during an experiment which may, depending upon the method, require several minutes to many hours." the company said.
The company said the Discovery X3 Differential Scanning Calorimeter generates three times the data as a standard DSC.
"The baseline quality and sensitivity of the Fusion Cell technology allows researchers to compare various formulations or competitive materials side-by-side under the exact same test conditions," TA Instruments said.
The company also introduced its new Trios-brand software feature, Batch Processing, which aligns "to assist with three times more data generated with the X3 DSC," according to the company.
Differential Scanning Calorimeters measure temperatures and heat flows associated with thermal transitions in a material, the firm said.
They are used for the "investigation, selection, comparison and end-use performance evaluation of materials in research, quality control and production applications," TA Instruments said. Tzero technology provides direct measurement of heat capacity.
The New Castle-based company also introduced a line of high-performance rheometers, including the Discovery Hybrid Rheometer HR 30.
"This new trio of high-performance rheometers are five times more sensitive than previous versions and offer class-leading versatility in a platform that makes it easier for users of all experience levels to obtain accurate rheological data," TA Instruments said.
The company enhanced its patented Magnetic Thrust Bearing, Optical Encoder Dual Reader and Advanced Drag Cup Motor technologies. Those moves provide "improved measurement sensitivity and dynamic data accuracy. Scientists are now empowered to measure weak intermolecular structures, lower viscosities, and obtain results on smaller volumes of low viscosity or weakly structured fluids, than previously possible," TA Instruments said.
That's a "critical consideration when working with scarce or novel materials," the firm said.
David Bohnsack, product manager for rheology at TA Instruments, said the firm's customers told them rheology is growing more important in their work.
"They need better data on a wider range of materials, and they have more people in their organizations making those measurements," he said in a statement. "The new Discovery Hybrid Rheometers support that need with better measurements, a system that is easier to use, and versatility to grow with their business."
Rheology studies flow and deformation of materials, the company said.
"Deformation and flow are referred to as strain or strain rate, respectively, and indicate the distance over which a body moves under the influence of an external force, or stress," according to TA Instruments. "For this reason, rheology is also considered to be the study of stress-strain relationships in materials."
"A rheometer is a precision instrument that contains the material of interest in a geometric configuration, controls the environment around it, and applies and measures wide ranges of stress, strain, and strain rate," it continued.
The company also introduced new software for users of the Discovery Hybrid Rheometer "of all levels of expertise."
The Trios-Express interface simplifies test set-up for users with little prior experience, the company said. A new AutoPilot feature also provides custom and guided interactions with the machine. This includes instructions, test methods, data analysis and reporting.
TA Instruments is a subsidiary of Waters Corp., a specialty measurement company with operations in 31 countries, including 15 manufacturing sites.


text:
"the Suspected player is picked out of the workout"
"The team player who is suspected of ill-treatment are taking a time out."
"the Player will not train or join the club's training camp, announces the club on its website."
"the Decision is taken in consultation with the player's family."
"An inexperienced player was arrested on Saturday by the on-call prosecutor, suspected of ill-treatment."
"He was released earlier in the day."
" We will continue the investigation and the investigative procedures that are left to him. I thought that the investigation can be carried out with him on the loose, the prosecutor said to Sportbladet."
"The club the club has been lying low in its comments on the player, but during Tuesday night, the club chose to comment on the situation on his website."
"the Player will have to take a timeout."
"In consultation with the player and the player's family, we have collectively agreed that the best thing for all parties is that he stays at home and does not participate in either the daily workout or another type of activity such as, for example, the upcoming training camp for the moment.”, writes the club on its website."
"”This is obviously a tough situation for all parties and the player's employers we would like to he now lands in situation and dialogue with his legal representatives in order to drive the process forward.”, write the club continued."
"The suspected abuse shall have taken place on Thursday, January 24."
"the Club also emphasizes that it will not comment on the incident or speculate on any repercussions, but let rättprocessen have its time."
"”Over the next 14 days so get the club the opportunity to get better insight into the situation and then make decisions in how we act in the future, depending of what emerges from the legal process.”, out of the club."

domain: news


text:
Reader Melissa asked a question about beta readers and online critique groups:
What’s the best time to start submitting work to a critique group? Should you wait until it’s finished or submit chapters as they’re written? Also, are beta readers the same as a critique group?
This made me think, as I frequently do, about the glory of critique groups and readers. All writers need them, no matter if you’ve never thought so or had unsuccessful situations in the past. Good critique partners and beta readers are worth their weight in gold, truly.
The Function of Online Critique Groups
The point of critique of any kind is to get other eyes (ideally, eyes that belong to writers who know what they’re talking about) on your work. How do you do that? First, there are critique partners or a critique group (the name isn’t important). These are other writers who you exchange work and commentary with, ideally on a regular basis. Some writers love working with local groups, but you don’t have to know your crit partners in person…these groups work well over the Internet or the phone, too.
When to Involve Writing Critique Groups
Critique partners or a writing critique group work best when writers convene regularly and are committed to one another, so that a core group can stay together over the course of many projects. Critique partners become intimately familiar with your writing, your stories, your strengths, weaknesses, and goals. This kind of continuity lets you get down to business and really get into the nitty gritty of the writing (ideally…follow the link to read more about what makes a great critique group or partner).
Some groups all want a writer to have a completed manuscript, others will be open to seeing work in progress…sometimes literally as it comes hot off the press (or printer). You should talk this over with your critique partners and make a decision that appeals to everyone.
It can be useful and exciting to get feedback on a project while you’re still creating it. It is even more useful, I think, to finish something, revise it on your own for a few passes, and then bring in your critique partners. A novel changes so much during the writing process, that it may be more helpful for you to work it out and get it written first, before even thinking about feedback and revision.
Writing Critique Groups vs Beta Readers
There is a small distinction, in my mind, between critique groups (or partners…I’ve used both terms interchangeably here) and beta readers. When I think of critique partners, I think of a group that meets regularly, will read whatever you give them, and really drill into it. That could mean reading an entire manuscript, or it could mean reading twenty versions of one chapter as you try to get it right. Critique groups should be very hands on and intense. Beta readers are people who read the whole manuscript and give feedback, but who may not give as much writing/revision/craft advice as your regular critique group. Beta readers are great if you want new eyes on a project, or if you want to hear from someone who doesn’t already know the story (like if you’re writing a murder mystery and really want to see if your red herrings work, but you’re worried whether your critique partners, who already know the whole story, will be able to judge after a few revisions).
Most of the professional writers I know have a regular critique group and then a few beta readers who they reach out to after the manuscript has been revised and polished, just for some quick feedback and a last minute read before it goes to their agent or editor, just to make sure the book is working.
For New Writers Looking for Critique Partners
But if you’re just starting out, don’t worry about having a pocketful of beta readers and a five-person bi-monthly, dedicated critique group. Maybe find a partner online (through the Verla Kay Blueboards, or the SCBWI) or at a writer’s conference. If that partner doesn’t work, find another one. I just did a Critique Connection post a few weeks ago, so that might be a place to search. The point is, start finding some critique opportunities and getting comfortable with the practice of giving, receiving, and incorporating feedback from a supportive community of writers (some constructive feedback examples here). Hone your revision skills. Take it one step at a time. You’ll find the right mix that works for you.
If your at a stage in your work where you’re ready to invest in an expert set of eyes, my book editing services will build on the work of your critique group.
20 Replies to “Beta Readers and Online Critique Groups”
Met my best individual critique partners through Verla’s. Can’t go wrong with the Blue Boards!
I heart my crit partners and beta readers. Writers are such a fantastic group, in general–generous with their time, willing to call you on your tics, and always rooting for you.
When developing a project, I like to get some initial feedback after the first 10k words I write to make sure my characters are compelling and the story has page-turning appeal.
There are different kids of crit partners and beta readers, too, and they all help catch different types of things. Couldn’t live without them. 🙂
Totally bizarre. I posted a poll this morning, over at Verla’s, about this! I was wondering about how many published/unpublished writers had critique groups/partners/neither. I was intrigued because I’d realised some writers don’t have critique groups/partners and I wondered about that. I would be lost without my crit group. It seems, though, that those who don’t rely instead on their editors or agents for crits.
I met my group and individual partners through Verla’s, too. What a difference it makes having that support and feedback! I know I’ve improved a lot faster than I ever would have on my own.
(I voted in your poll, Siski 😉 )
I met my crit groups (I have two – one for PBs and one for MG/YA) through the SCBWI, and they are awesome. I’d be lost without them! And I found one CP through your critique connection, Mary, so thank you so much for that!
This was a great post; I appreciated reading your distinction between CPs and Betas. They perform such different functions, I can’t imagine how a writer can go without either of them.
I’ve learned so much from my critique group partners and have now come to the point where I feel I have good feedback to offer as well. It’s wonderful to see others stories growing and advancing through your feedback and their hard work and dedication.
For me, I like to have readers while I’m writing the first draft who are excited about the story, claim (however truthfully) to enjoy the story, demand more chapters, etc. It’s a big love fest, with very little (if any) critique involved. It keeps me excited about the manuscript and keeps me from second-guessing myself.
The second I finish the last chapter, though, I stop wanting the happy love-fest readers and start needing people who are willing to help me rip the thing apart. I’ll usually revise it once on my own and then send it off to a first round of critiquers. Then repeat as necessary (or ad nauseum) until the book is ready.
Most of my critique partner experiences have been wonderful. Even the time I made a complete fool of myself with my poor writing, and I drove home in tears, saying, “I’ll show them.”
I’ve grown a bunch since that time over thirteen years ago. So, I say that’s wonderful.
Nice post, Mary!
I don’t know what I’d do without my critique partner or my beta readers! Not only are they essential to my writing, but they’ve become really great friends too.
Luckily I’ve had mostly positive critique/beta experiences. There are some critique feedback that can be harmful to a writer’s tenacious spirit to improve so I do caution to pick your critique/beta’s wisely. Exchanging a chapter or two first sometimes can give you a feel for the critique partner’s style.
Great post, Mary! It was a nice reminder of the importance of the opinions of others. For me, the feedback of other people is truly the key to improving my ideas, tweaking my plot, inserting details as necessary, and tightening up loose ends. It’s the ultimate one-stop-shop for story improvement! Except for the extensive revisions afterward, of course. 😉 But it’s not so bad when you know what changes people are looking for.
I established my own monthly writer’s group, and we’re now nearly ten members strong, nine months later. Since starting the group, I’ve noticed significant changes in my style and the strength of my writing. The advice and opinions of another set of eyes are truly invaluable. They pick up on things you didn’t, provide honest opinions you [think] you don’t want to hear, and ultimately help your writing. As long as I am a writer, I’ll always have my critique group.
Thanks for this post. I will be meeting with my critique tomorrow, and they are invaluable. I believe the most significant thing I’ve learned from our ten years together is how let go of being defensive about my writing. I may not always be delighted to hear their comments, questions or suggestions. But I trust they are sincerely offered with the goal of helping me make the best book I can. When I’m not feeling anxious or defensive I am much more able to truly hear what they have to say. Once I’m able to listen openly, I am more able to take suggestions I think will help me make the book I’m trying to make. And, their comments that don’t match my goal and vision of the book can be placed aside. My group has also developed a way to group-think-aloud. By tossing ideas and questions back and forth we often find just the right tweak or word or sentence needed in the manuscript being discussed.
There have certainly been times I left my writing group feeling flat or disappointed they didn’t award my new story the Noble prize. But with only one exception, by the time I reach home I have always excited to get back to work on my manuscript. Why? My friends’ comments, questions, and challenges are invigorating.
Having a critique group has got to be one of the most important things to do as a writer. I’ve had some great one-off critiques from Verla’s, and made some lovely friends from there too.
My crit group was made up of WriteonCon folks and they are awesome. I have been a beta reader for a couple of people recently even though they already have crit groups and it is really good giving a different perspective, new ideas when you haven’t read the ms thousands of times.
Great post Mary!
I have three separate critique partners now, thanks to a hookup Mary did a month or so ago, and its been really interesting to see how differently these individuals respond to the same material. It’s been invaluable. They’re looking at a first draft of a first (unfinished) novel, and I’ve been moved to make major changes: collapsing characters, re-plotting whole sections, shuffling chapters — even parts of the book they haven’t seen yet! Having critique partners fairly early in the writing process has saved me from traveling a long way down dead end roads, I think, even if I’m making intuitive leaps rather than changes based directly on their suggestions (although there are certainly some of those too). Thanks to these crit partners and workshops at Big Sur (truly inspiring!) I’ve grown tremendously as a writer — I highly recommend both.
Thanks for the response, Mary. I found a crit partner through Verla Kay and it has been great! Her novel is complete and mine is still being written, but we figured out an exchange pattern that works for us and I couldn’t be happier. That’s not true–I will be happier when I finish my darn book! 🙂
I found my critique partner through your critique connection and she’s AMAZING. My ms is so much stronger now with her help. I kept meaning to post a thank you on here, and forgetting to do so, so here it is.
THANK YOU. For the connection and for the ongoing advice!
Thanks for this post. I have often wondered when would be the best time to share work with a critique group and now I am sure that after the second draft would be best.
Tried to clock through to “Critique Connection” and the link is broken.

domain: literature


About Tyler Shaw
Singer Tyler Shaw’s slick pop has landed him several hits in his native Canada, including 2012’s “Kiss Goodnight”.
• The musical streak in Shaw’s family goes back generations. His great-grandparents played in one of the only wedding bands in Saskatchewan, Canada. Nevertheless, Tyler wanted to be a pro soccer player before picking up the guitar.
• After winning a singing competition sponsored by the Canadian cable channel MuchMusic, Shaw signed with Sony Music Canada and released his debut single, “Kiss Goodnight,” in 2012. The track peaked at No. 24 on the Canadian Billboard Hot 100 en route to going platinum.
• In 2014, he earned a Juno nomination for Breakthrough Artist of the Year.
• Shaw’s 2015 debut album, Yesterday, spawned the hits “House of Cards” and “Wicked,” both of which went gold in Canada.
• Intuition, Shaw’s 2018 sophomore album, yielded three hit singles: “Cautious,” “With You,” and “To the Man Who Let Her Go.” The album brought Shaw a Juno nomination for Pop Album of the Year.


Kenny Electric worked with Wood Group and Black Hills Energy to bring the Corriedale Wind Farm to life. There were over 80,000 feet of fiber optic cable pulled for the twenty-one wind turbines and substation SCADA panels that Kenny connected. Our team provided fiber terminations and Optical Time Domain Reflectometers (OTDR) which are used to verify the performance of new fiber optics links and to detect any complications.
With more than 60K wind turbines across the United States, wind power has become the third-largest source of electricity generation in the nation. The collective capacity of those wind turbines can power up to 34 million homes. Wind remains is a critical part of the Energy Department’s strategy to cut carbon pollution and bring the next generation of clean energy to fruition.


President Donald Trump has accused the Democratic Party of trying to rig the 2020 US presidential election in favour of candidate Joe Biden.
Trump asserts that in Wisconsin, Georgia, and elsewhere, he won the states “by a lot,” but that his victory is now being “whittled down.”
“We’ll not allow the corruption to steal the election,” he says. Results are being manufactured.
He repeats his claim that Democrats are “trying to steal an election, they’re trying to rig an election.”
“Democrats never believed they could win the election honestly,” he says, falsely claiming that millions of ballots for Biden have been “sent out.”
He says, without evidence, that election officials are part of a “corrupt Democrat machine” and are accepting votes without requiring “any identification whatsoever.”
He claims election officials in states such as Pennsylvania are “trying obviously to commit fraud” and keeping observers from overseeing the tabulation process.
Trump says people who are being banned from observing the vote count “are very unhappy and become somewhat violent.”


text:
Buttigieg Vows Taxpayer-Funded 'Free' Health Care for Illegal Immigrants
Democrat presidential candidate pledges to open up Obamacare to illegal aliens
Democrat presidential candidate Pete Buttigieg has vowed to give illegal aliens access to "free" health care by opening up taxpayer-funded Obamacare to undocumented immigrants.
While speaking to an illegal immigrant on Sunday, former South Bend Mayor Buttigieg promised they would have taxpayer-funded health care if he was elected president.
“As you know, the Affordable Care Act, one of the many missing pieces that it has is that the exchanges are not available to the undocumented,” Buttigieg told the illegal alien.
“I would change that, and that would be a change that would come with the ‘Medicare-for-All-Who-Want-It’ plan that I am proposing.”
Buttigieg made the pledge to open-up taxpayer-funded health care for illegals during a political exchange with Planned Parenthood in Nevada on Sunday.
The Democratic White House hopeful told the illegal immigrant that he viewed her being no less American than any U.S. citizen, despite her illegal status.
“So, first of all, this should go without saying but it’s important to say out loud that I regard you and all DACA recipients as American as I am or anybody else is in this room,” Buttigieg said.
In December, Buttigieg also expressed his desire that he would open up taxpayer-funded healthcare to illegals during a conversation with a voter in Spanish.
“So the most important thing for me is that we offer the opportunity for health care to all in our country, and this includes the opportunity to buy this plan of Medicare-for-All-who-want-it,” Buttigieg said, according to a translation of his remarks.
“That is our solution," he added.
"And this opportunity to buy this plan is for everyone regardless of their immigration status.”
A clip of the exchange was published by The Republican National Committee Research Team.
“I’ve said it before, and I’ll say it again. Just because Pete Buttigieg is from Indiana doesn’t make him a moderate,” Republican National Chairwoman Ronna McDaniel said on Twitter.
Last week, Buttigieg caused outrage while promoting a new "visa program."
As Neon Nettle previously reported, Buttigieg's program aims to encourage legal immigrants to flood small American towns in order to increase the regions’ populations.
The program aims to benefit corporate investors, big business and housing developers, while seemingly sidling ordinary residents.
Buttigieg touted his 'investor-friendly' plan during a campaign stop in Merrimack, New Hampshire, which would overwhelm small American towns with legal immigration to drive population growth.
Buttigieg told the crowd:
I’m proposing what we call “Community Renewal Visas” that when a community that is very much in need of growing its population recognizes that, and makes a choice to welcome more than its share of new Americans that we create a fast-track if they apply for an allotment of visas, that goes to those who are willing to be in those areas that maybe are hurting for population but have great potential.


text:
Statement from Commissioner Daines on Accreditation Requirement for Office-Based Surgery Practices
ALBANY, NY (July 14, 2009) – Today all New York State physicians' offices performing invasive procedures and surgery requiring moderate or deep sedation must be accredited, as required by New York State Law. Under Governor Paterson's leadership, the Department of Health has been working diligently to ensure patient safety. Accreditation will help assure that New Yorkers receive the highest quality of care when undergoing surgery in outpatient settings.
Currently, there are approximately 500 New York State providers that are accredited to perform office-based surgeries and 200 more are awaiting accreditation. Any provider that has not received final accreditation as of yesterday, July 13, cannot perform office-based surgeries requiring moderate or deep sedation or general anesthesia today.
This requirement is mandated by the Office-based Surgery Law enacted in 2007. As of today, only accredited physicians' offices may perform office-based surgeries requiring moderate or deep sedation or general anesthesia in New York State. Physicians, physician assistants, and specialist assistants who continue to perform specific procedures in a non-accredited setting may face professional misconduct charges.
New Yorkers will now be able to check a listing of accredited office-based surgery practices by county on the state Health Department Web site at: http://www.nyhealth.gov/professionals/office-based_surgery/practices/.

domain: law