text:
Plain English Summary
Background and study aims
It is common practice to treat patients with a heart attack affecting much of the heart muscle, known as an ST-segment elevation myocardial infarction (STEMI), with percutaneous coronary intervention (PCI). PCI for STEMI can be either primary PCI or rescue PCI or a pharmacoinvasive strategy. Doctors trained to carry out this procedure are called interventionists. Interventionists identify the 'culprit' lesion, which is the blocked coronary artery responsible for the heart attack, clear any blood clot, and usually site a small device known as a stent in the artery to keep it open and allow the blood to flow freely to the heart muscle (revascularisation). The amount of disease varies from patient to patient. Some patients will have remaining narrowing in arteries that have not actually caused the heart attack. These narrowings are referred to as 'non-culprit lesions'. Following PCI, some interventionists will treat these patients with medical therapy and only if the patients come with worsening symptoms will consider looking at the arteries again. However, other interventionists, following primary PCI, will treat the patient with medication, and in addition, plan to bring the patient back to the lab in the near future to treat the non-culprit lesions ('staged PCI'), which may minimise the risk of further heart disease. This study is looking at both of the treatment strategies described above to find out whether a strategy of complete revascularisation will be better than a strategy of culprit-lesion-only revascularisation.
Who can participate?
Patients who have undergone primary PCI will be invited to participate in the study within 72 hours of their procedure.
What does the study involve?
Patients are randomly allocated to one of two treatment groups: complete revascularisation or culprit-only revascularisation. Those allocated to complete revascularisation will undergo staged PCI, within 45 days, of all suitable non-culprit lesions. Those allocated to undergo the culprit-only revascularisation will be treated if they have worsening symptoms in future.
What are the possible benefits and risks of participating?
All patients will receive a very high level of treatment and follow-up by trained doctors. You will be followed carefully at regular intervals by the research team involved in the study. Your medications will be reviewed at regular intervals and you will also have your cholesterol levels and blood pressure checked. An additional PCI procedure carries a small risk (about 1 in 100) of procedure-related complications such as heart attack, stroke or death. This risk will be higher or lower in some patients, depending on their age and other factors. In addition, there is a small risk of the contrast dye affecting the kidneys (less than 1 in 100) and allergic reaction to the contrast dye (less than 1 in 100). This study is being conducted to find out if an additional PCI procedure will lower your long-term risk of subsequent heart attack or death. The additional PCI procedure you might receive will give you a dose of ionising radiation equivalent to about 2 years average natural background radiation. The Health Protection Agency (now Public Health England) Radiation Protection Division describe a radiation exposure equivalent to a few years average natural background radiation as Low Risk. For some patients the second procedure would not be additional, and hence no additional risk.
Where is the study run from?
Sheffield Teaching Hospitals NHS Foundation Trust is the UK lead site.
When is the study starting and how long is it expected to run for?
The study started recruitment in the UK in January 2013 and will run for 5 years.
Who is funding the study?
1. Astra Zeneca (Canada)
2. Boston Scientific (USA)
3. Population Health Research Institute (Canada)
Who is the main contact?
Population Health Research Institute (PHRI)
Trial website
Contact information
Primary contact
Ms Emilija Makaji
Contact details
Population Health Research Institute
Additional identifiers
EudraCT number
ClinicalTrials.gov number
Protocol/serial number
Study information
Scientific title
A randomised, comparative effectiveness study of COMPLETE versus culprit-only revascularization strategies to treat multi-vessel disease after primary percutaneous coronary interventions (PCI) for ST-segment elevation myocardial infarction
Study hypothesis
The study is designed to determine whether, on a background of optimal medical therapy with low dose aspirin and ticagrelor, a strategy of multivessel revascularisation, involving staged PCI using drug eluting stents of all suitable non infarct related artery lesions, is superior to a strategy of culprit lesion only revascularisation in reducing the composite outcome of cardiovascular (CV) death or new Myocardial Infarction (MI) in patients with multivessel disease who have undergone successful culprit lesion primary PCI for STEMI.
Primary hypothesis: A strategy of complete revascularization will be superior to a strategy of culprit lesion only revascularization in reducing major clinical outcomes including the composite of CV death or new MI and the composite of death, new MI or ischemia-driven revascularization
Secondary hypothesis: A strategy of complete revascularization will lead to a better quality of life as measured by the EQ-5D and an improvement in angina control as measured by the Seattle Angina Questionnaire compared with a strategy of culprit lesion only revascularization.
Ethics approval
Medical Research Ethics Committee, 07/10/2013, 13/EM/0343
Study design
Randomised; Interventional; Design type: Process of Care
Primary study design
Secondary study design
Randomised controlled trial
Trial setting
Trial type
Patient information sheet
Not available in web format, please use the contact details below to request a patient information sheet
Topic: Cardiovascular; Subtopic: Cardiovascular (all Subtopics); Disease: Cardiovascular
1. Complete Revascularisation: Staged PCI using second generation drug eluting stents of all suitable non-culprit lesions.
2. Culprit Only Revascularisation, Culprit Lesion-Only Revascularisation: no further revascularisation of non-culprit lesions.
This will be a randomised, comparative effectiveness study of complete versus culprit-only revascularisation strategies to treat multi-vessel disease after primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI).
Patients, over the age of 18 years of age, who have undergone index PCI for STEMI and have at least one other non-culprit lesion in a vessel that is greater than or equal to 2.5 mm in diameter with at least 70% diameter stenosis (on visual estimation) or 50-69% stenosis with fractional flow reserve (FFR) less than or equal to 0.80, will be eligible to participate in the COMPLETE study. Index PCI for STEMI can be either primary PCI or rescue PCI for failed fibrinolysis or a pharmacoinvasive strategy where PCI is performed routinely 3-12 hours after initiation of fibrinolysis.
The direct clinical care team performing the primary PCI will be aware of the patient's suitability. The clinical care team, if the clinical situation allows, will ask the patient if they would be interested in helping with the study. The clinical care team will alert the research team, who will approach the patient and provide sufficient information for the patient to decide whether to participate in the study. If it is not appropriate, for clinical reasons, to discuss the study at the procedure, the interventionist or a member of the clinical care team may ask the patient following the procedure and when the patient is well enough. The research team can confirm the patient's suitability by accessing the database completed by the interventionist following the primary PCI. The study will be explained, in depth, to the patient by a research team member who will satisfactorily answer any questions that may arise and give the patient sufficient time to decide whether to participate in the study.
When written informed consent has been given by the patient, the research team will collect details of demographics, clinical assessment, medical history, concomitant medications, smoking status, blood pressure, lipid results including low-density lipoprotein (LDL) cholesterol, and glycaemic status (HbA1c). The research team will also assess Canadian Cardiovascular Society (CCS) classification, a measure of chest pain, and NYHA classification, a measure of heart failure severity, as well as ask the participant to complete a quality-of-life assessment.
Within 72 hours of the index PCI, the patient is randomised into one of the two study arms: complete revascularisation with background optimum medical therapy, or culprit-lesion-only revascularisation with background optimum medical therapy. Some of the assessments cited above are then repeated.
For those patients randomised to the complete revascularisation arm of the study, the staged PCI must be performed within 45 days. An electrocardiogram (ECG) is done within 12 hours prior to the procedure and 24 hours post procedure (or hospital discharge if earlier). At this time point, blood is drawn for cardiac markers and clinical outcomes and medical therapy is noted.
At hospital discharge, clinical outcomes, medical therapy, smoking status and CCS and NYHA classifications are documented. Six weeks into the study (plus or minus 14 days), the same information is collected again. This visit may be done either in a clinic or over the telephone.
At 6 months, a clinic visit (or optional telephone contact) is performed where the clinical outcomes, medical therapy, smoking status, serum LDL cholesterol, blood pressure, glycaemic status, quality of life assessments, and CCS and NYHA classifications are collected. Thereafter annual visits at 1,2,3 and 4 years are completed in the clinic or via telephone contact with documentation of clinical outcomes, medical therapy, smoking status, and CCS and NYHA classifications. The final visit/year 5 is a repeat of the month 6 visit, without the option of telephone contact.
Intervention type
Not Applicable
Drug names
Primary outcome measure
Cardiovascular (CV) death or new MI; Timepoint(s): hospital discharge, 30 days, 6 months, 12 months and then annually for up to 5 years
Secondary outcome measures
1. To determine whether complete revascularisation reduces the composite of cardiovascular (CV) death, new Myocardial Infarction (MI) or ischaemia-driven revascularisation.
2. To determine whether the initial strategy of complete revascularisation improves angina control, as assessed by the Seattle Angina Questionnaire (SAQ) Frequency Scale, and health-related quality of life scale at 6 months and 5 years/final follow-up compared to baseline.
To determine whether an initial strategy of complete revascularisation is superior to an initial strategy of culprit lesion only revascularisation in reducing the composite of CV death, new MI, ischaemia-driven revascularisation or rehospitalisation for unstable angina or hospitalisation for heart failure and each component of the key secondary objectives taken separately as well as all-cause mortality, stroke, stent thrombosis, major bleeding, economic evaluation, including health resource utilization, costs and cost-effectiveness.
Overall trial start date
Overall trial end date
Reason abandoned (if study stopped)
Participant inclusion criteria
1. Men and women within 72 hours after successful PCI (preferably using a drug-eluting stent) to the culprit lesion for STEMI. PCI for STEMI can be either primary PCI or rescue PCI for failed fibrinolysis or a pharmacoinvasive strategy where PCI is performed routinely 3-12 hours after initiation of fibrinolysis
2. Multi-vessel disease defined as at least 1 additional non-infarct related coronary artery lesion that is at least 2.5 mm in diameter that has not been stented as part of the primary PCI and that is amenable to successful treatment with PCI and has
2.1. at least 70% diameter stenosis (visual estimation) or
2.1. at least 50% diameter stenosis (visual estimation) with fractional flow reserve less than or equal to 0.80
Participant type
Age group
Target number of participants
Planned Sample Size: 3900; UK Sample Size: 100
Participant exclusion criteria
1. Planned revascularisation of non-culprit lesion(s)
2. Planned surgical revascularisation
3. Non-cardiovascular co-morbidity reducing life expectancy to less than 5 years
4. Any factor precluding 5 year follow up.
5. Prior coronary artery bypass graft (CABG) surgery.
Recruitment start date
Recruitment end date
Countries of recruitment
Australia, Brazil, Bulgaria, Canada, China, Czech Republic, Denmark, Finland, Germany, Greece, Hungary, Israel, Italy, Lithuania, Macedonia, Mexico, Netherlands, Poland, Romania, Russian Federation, Serbia, Spain, Sweden, Ukraine, United Kingdom, United States of America
Trial participating centre
Population Health Research Institute, Canada
Hamilton, ON
Funder type
Funder name
Astra Zeneca (Canada)
Alternative name(s)
Funding Body Type
Funding Body Subtype
Funder name
Boston Scientific (USA)
Alternative name(s)
Funding Body Type
Funding Body Subtype
Funder name
Population Health Research Institute (Canada)
Alternative name(s)
Funding Body Type
Funding Body Subtype
Results and Publications
Publication and dissemination plan
Not provided at time of registration
Intention to publish date
Participant level data
Not provided at time of registration
Basic results (scientific)
Publication list


text:
Publisher : Center for Responsive Schools, Inc.
Release : 2007
Language : En, Es, Fr & De
Book Description :
Written with warmth and humor, Yardsticks offers clear descriptions of children's development. This comprehensive, user-friendly reference helps teachers and administrators use knowledge of child development to shape classrooms and schools where all children can succeed. For each age, this book includes: Narrative description of developmental traits Charts summarizing physical, social, language, and cognitive growth patterns Suggestions for curricular areas: reading, writing, mathematics, and thematic units Favorite books for different ages. What's new in the third edition: A new, brief overview of issues in the development of bilingualism and biliteracy among Latino/Hispanic children A new appendix on the "birthday cluster exercise" for applying the information in the book to working with a whole class of students An updated list of recommended children's books An updated list of recommended resources for teachers and parents.

subset: commoncrawl


text:
Marketing Plan NOW - The Tools
There is a hierarchy of content between information, know-how, and learning. Therefore, tools
support learning.
The marketing plan tools we offer consist of helpful materials such as videos, presentations,
articles, a home-made marketing plan glossary and a few related links.
A sample of our marketing tools: A 4 min. 32 sec. introductory
approach by Danny Abramovich to key oncepts: Management,
business planning process, business plan and marketing plan.


text: Bushranger Exhaust Jack - 72X04 The best 4WD jack in sand, snow, mud or on soft sloping ground. Simple to use and highly efficient, with no complicated moving parts to jam or seize. Features a reliable 1 way valve at the end of the hose, which will keep the bag inflated for hours. Great to lift your vehicle out of a hole when it's up to the axles, whether in snow, dirt, mud or sand. Comes as a complete kit including two protective mats to prevent puncturing the bag, a pair of work gloves to keep your hands clean and protected, a puncture repair kit in case the bag is pierced, laminated operating instructions and a handy carry bag in which you can store everything. Features: Suits medium to large 4WD Wagons and Utes (including long travel suspension vehicles) *Not for F250's Exhaust Jack - 75cm lift Maximum Vehicle GVM - 4 Tonne.


text:
I needed a stand for a wine chiller. Wine fridge? Wine cooler? Bartles and Jaymes?
Complete shot first:
I had this extra bed headboard (and tailboard? is that a thing?). It's from IKEA. It's made of wood. I hate throwing
good wood away. I'll burn/crush/destroy and otherwise exact lament upon particle board furniture all day long but in its original native tree form I have an affinity with it; I want to see it live anew; this respect is akin to a steak versus chicken nuggets.
Enter bedframe/headboard/tailboard/woods:
I cut it down so the legs were all equal and gave them a staining.
I then cut the thin back board and linked them together with some dowels and wood glue to give it some rigidity and to get it to the right size.
I used some spare wood I had lying around as a topper for it and snapped up a segment of 1×2 (these not shown) to the back as a brace.
Voila: recycled.


text:
Dungeon Contractor: I Know Why The Cave Cricket Sings
The most underused sense in gaming is smell. This is not unnatural. The human axis of experience does not revolve around smell. Smell is the background music of our lives, never the action taking place on the screen. A particular smell may send us towards memory or thought, much the way different musical themes will do the same thing in a movie or opera. Consequently, this is what makes it so hard to implement in a game. In real life smell goes unnoticed so often that it only is noticed if there is a darn good reason for it to be. Bringing it up in a game can seem unnatural.
The most underused sense in gaming is smell. This is not unnatural. The human axis of experience does not revolve around smell. Smell is the background music of our lives, never the action taking place on the screen. A particular smell may send us towards memory or thought, much the way different musical themes will do the same thing in a movie or opera. Consequently, this is what makes it so hard to implement in a game. In real life smell goes unnoticed so often that it only is noticed if there is a darn good reason for it to be. Bringing it up in a game can seem unnatural.
Mind you, we are not discussing the most unused sense in games, which is probably taste, but taste works on a different sort of angle. Generally taste does not get forced upon someone like smell does. In fact, this article is not even actually about smell, but smell as a part of a class of things that go along with any dungeon.
Last time I explained the importance of dimension. This time it is the importance of reality. Reality has been a fickle topic in the Dungeon Contractor series, but that should not be much of a surprise to any student of reality. What reality means or is changes on a frequent basis, depending on needs and circumstances, and in this circumstance reality is a crutch.
I've always had a special place in my heart for that saying.
Reality is a crutch for dungeon design. It is not the thing, it is not like the thing, but it exists for the sake of helping the thing do its own thing. Is smell a crutch as well? No, but smell is a part of a class of crutch items that all derive from reality. Mistake them for the thing itself and design will wander way off.
What things am I referring to? When's the last time you were in a dungeon? (See 1, below) It is nearly impossible to get to a dungeon nowadays, not because they never existed but because they need to be preserved and reconstructed. But even then, past the faux murals and the environmental controls there is often a residing feeling of unpleasantness. It must be sought for elsewhere in reality for most people: the humidity of an unfinished basement you look for the blown fuse in armed only with an oil lamp, the moose carcass you have to hide from the English soldiers in, the caving trip you took, back when you were of an age adults deemed it proper to take you places like caves, the pedestrian viaduct from Michigan Avenue to Oak Street Beach, at least back in the late eighties and early nineties.
In all of these, and as many other as people would care to cite, there are often smells that accompany them. Yes, in games we forget to bring up those smells, but more to the point is that none of those smells are anything pleasant. For an evolutionary explanation, our ancestors who survived were not the ones who thought mold had a nice bouquet, because they all died of vitamin D deficiencies. There is nothing pleasant about any of the environmental experiences of a dungeon. The cave cricket sings to annoy you.
The list of possible environmental ills you could throw at characters is formidable. Frankly, such a list is also better suited for a series discussing run-time issues as opposed to design. The design lesson to be learned is to remember the unpleasantness.
It does not need to be included, or rather, do not find the need to discern which corners have been pissed in how often because you need a smell map to go along with the regular one. If such a map fits with your concept, more power to you. Do keep in the back of your head this place of adventure is not pleasant without some serious remodeling. No one but a creature designed to be there is going to want to be there. I have equated dungeons to fun houses in the past, but these fun houses are a little more sophisticated. Perhaps they are the deserted place down the block that all the kids say is haunted; but most likely not, because there are real yet cartoonish enemies who jump out from the shadows. No, it is a superlative fun house, the kind you laugh as you exit but quietly vow never to go back to again. Keeping a general feel of nastiness in the back of your head will set the tone better than any number of glowing fungi.
Admittedly this time has been discussing dungeons in specific, as opposed to the concept of dungeon in general, and so this is not an idea that can be consistently applied. On the other hand, it is typical the place is supposed to be uncongenial, and it goes without saying that understanding the default theme for a place helps to square away whatever other themes are being laid on top.
Next time: where do those phosphorescent plants come from?
1 - The author would like to take this moment to point out he once went crawling through the tunnels under the Temple of Apollo at Delphi - with a candle stuck in his mouth nevertheless - not because it was especially dungeon-like but because he wanted to brag.
- Login to post comments
I like the fun-house explanation of dungeon design. . . .
Another word on smells: I've toyed with the idea (but never gone through with it) of fishing up sample smells in zip bags for release at appropriate times in a game: rotted wood in a damp dungeon chamber, dry soil for a drier area, sawdust for a stable (just sawdust, thanks. . .), ammonia for a green dragon, etc.
Smells _do_ evoke things nicely and involuntary, but I never actually went through with my plan.
- Login to post comments


text:
Yes, this is what I had in mind. Thanks. One further question though . . . > * In WSDL, an interface is always a different thing > from a binding > (even if they have the same name), and they will stay > different > when mapped from a WSDL file to RDF. In the ontology, however, > one can assert that one resource is both an interface and a > binding and this introduces no RDF-level inconsistency. Doesn't Arthur's QName --> URI mapping algorithm prevent this? David BoothReceived on Monday, 6 February 2006 20:42:33 UTC
This archive was generated by hypermail 2.4.0 : Friday, 17 January 2020 23:06:56 UTC


text:
On August 28, 1963, during "the March on Washington," Rev. Martin Luther's King, Jr. gave his famous "I Have a Dream" speech on the steps of the Lincoln Memorial. On that same day, just 45 miles away, the practice of segregation was discontinued at the Gwynn Oak Amusement Park just outside of Baltimore. And an eleven-month-old baby named Sharon Langley was the first African American child to go on a ride there when, along with two white children, she rode on the park's classic, old-time carousel.
The next day, "amid all the news stories about the March on Washington, there were also stories on Sharon Langley's merry-go-round ride. Three kids – one black and two white – riding together provided an example of the harmony King spoke about at the march, when he hoped that one day black children and white children would regard each other as "sisters and brothers."
On today's lunchtime bike ride I went see that carousel. But I didn't have to ride all the way to Baltimore to do so. Today that very same carousel is here in D.C., on the National Mall (MAP) in front of the Smithsonian's Arts and Industries Building, where young children enjoy themselves while their parents watch them ride the seemingly benign carousel, unaware that it has a rich history which is much more interesting than its appearance would suggest.
On April 12, 1967, the Smithsonian Secretary S. Dillon Ripley opened the carousel on the National Mall. The original carousel was built in 1922 by the Allan Herschell Company, and was accompanied by a 153 Wurlitzer Band Organ. At that time, rides cost 25 cents. However, not everyone was happy to see a carousel placed on "America's front yard." Some were concerned that that the carousel, along with the popcorn wagons and some outdoor puppet and musical performances that were already there at the time, would lead to the Smithsonian developing into what the New York Times termed "an ivy-covered Disneyland." But that never happened, and the carousel remains to this day.
Today's carousel is not the original, though. Due to wear and tear the original carousel was replaced in 1981 with the carousel from the Gwynn Oak Amusement Park, which was forced to close in 1973 after suffering severe damage from flooding when Hurricane Agnes. The Gwynn Oak carousel is 10 feet larger in diameter and has 60 brightly-painted horses, as opposed to the former which had 33. It also has a few non-moving seats, and one sea dragon. And riding on the carousel is not limited to children. All are welcome, including adults, as long as you're willing to pay the current ticket price of $3.50.
[Click on the photos to view the full-size versions]


text:
Chevron Annex Featured in Green Building and Design
25 Feb 2014 by Chris Martin
Green Building and Design features the Chevron Annex in their January/February 2014 publication. The article recounts how this robust laboratory evolved into a thoroughly sustainable facility through an air-rights concept. Check out the Green Building and Design article Up in the Air: Chemical Reactions, with no room to grow outward, Wilson Architects expands a laboratory […]Read Post
Discussion Board • Sustainability Drivers
20 Feb 2014 by Lisa Hellmuth
In their most recent publication, Green Building and Design contacted our in-house sustainability leader, Kristine Renner, to participate in a discussion on the driving force behind the sustainability movement. In this discussion board, Kristine Renner responds along with other sustainability gurus, to the question: "Are we at a point where profits, rather than incentives, can drive […]Read Post
WA Promotion • Jacob Werner
01 Feb 2014 by Lisa Hellmuth
Wilson Architects named Jacob Werner, AIA, LEED AP BD+C an Associate of the firm. Jacob joined WA in 2012, and specializes in the planning, design, and construction of laboratory buildings. He successfully guides teams through collaborative listening and attention to detail. Jacob has extensive experience with sustainable lab planning and design, including a deep understanding […]Read Post


text:
SMACS Multi-Entry is a secure interlock solution to prevent tailgating whilst enabling efficient throughput. SMACS Multi-Entry is ideal for busy building entrances or access to conference rooms. SMACS Multi-Entry can be configured to provide different levels of security, day or night.
Main Features
- Allows the transit of up to 20 people per minute
- Enables the passage of up to 6 people in a single transit
- Easy to install and retrofit
- Suitable for wheelchair access
SMACS Multi-Entry is installed within a 2-door airlock and provides secure anti-tailgating where there is the need to have multiple users accessing the secured area in a single transit. SMACS Multi-Entry will allow access to multiple users in to the scanning area on presentation of authorised ID. Should a user fail to present an authorised ID, SMACS Multi-Entry will trigger an alarm signal and alert the building's security.
Processing Unit Dimensions
- 240 x 165 x 70 mm
Cabinet Dimension (Optional)
- 600 x 600 x 210 mm
- Standalone
Hardware Interface
- Digital I/O, dry contacts
Sensors Compatibility
- Analog, thermal, laser, IR
Guidance (Optional)
- Audio-Visual
Maximum Airlock Size
- Unlimited (sufficient cameras required)
Ceiling Height
- 2.50 m - 4m
Access Control Interface
- Wiegand, dry contact
Alarm Interface
- Digital I/O, dry contacts
Power Supply
- 110-230Vac 240W
Size of Singularisation Zone
- 1.5 m x 1.5 m
Optional Security Processes for Tailgating Detection
- Timed modes
- Customisable mode – one way or bi-directional control
- Thermal imaging camera
- Additional internal cameras


text:
Science for Excellence : Physical Science: Level 4
Chambers, Paul and Jeffrey, Stephen (2011) Science for Excellence : Physical Science: Level 4. Hodder Gibson, Paisley. ISBN 9781444145212Full text not available in this repository.
Science for Excellence Level 4: Physical Science is directed towards the Level Four Science experiences and outcomes of Curriculum for Excellence in Scotland. Its main focus is on those relating to the Forces, Electricity and Waves organiser, but it also encompasses a number of the outcomes in the Planet Earth organiser. It was felt that much could be addressed in this section from a physical science perspective that is different to the traditional recall of factual content regarding the Solar System. It makes frequent reference to key concepts identified in Curriculum for Excellence and the topics chosen in the text can be linked with content across the other organisers. Although the chapters are designed to meet Curriculum for Excellence Level Four outcomes, their approach and content have also been influenced by the need to articulate with National Four and National Five developments which were underway at the time of writing. An enquiry-based approach suggests activities which are designed to encourage pupils to plan and design experiments which present opportunities for individual investigation or practical challenges but also others which are more designed to provide progression onto the next stage. This is reflected in the inclusion of content covering forces, gas laws, sound and electricity. In an attempt to allow our pupils to make more informed decisions on scientific issues relating to their own experiences, the chapters have a strong Scottish viewpoint, but this is balanced by reference to global issues. It is hoped that the physical science content, presented alongside examples of historical ideas, will develop pupils' awareness of science as a continuing process involving tentative ideas, and that what is considered correct at one time may be refined in light of new discoveries. Some of the activities in the book involve experiments. These should only be attempted under the instruction of the Science Teacher and in accordance with the appropriate safety guidelines. Problems and activities are designed to examine and extend the content of the chapters. Skills in literacy and numeracy as well as an awareness of the importance of health and wellbeing will be developed through these exercises – look out for the icons shown at the end of this Introduction. Some chapters allow for numerical and graphical activities where others seek to reinforce the scientific principles contained in the main text. It was also felt that in an attempt to make the learners more active participants, open ended and pupil investigation activities should feature. These activities encourage individual project work, research and group work, with learners being asked to make informed decisions on scientific advances which may have ethical or societal implications. The tasks are designed around the 'broad features of assessment in science'. The principles and practices outlined in Curriculum for Excellence have been adopted throughout the Science for Excellence series. The series is designed to be used in conjunction with schemes of work which reflect learning and teaching approaches which are most applicable to the sciences. The series provides opportunities for scientific enquiry and examples of scientific scenarios where pupils can, for example, link variables to determine relationships or improve their scientific thinking or make informed judgements on the basis of scientific principles. Scientifically Literate Citizens The series' use of real data and experimental type situations is designed to support the development of pupils' scientific attitudes. They will be able to look at data critically, make informed judgements on the basis of these and be critical and analytical of the science as well as the implications of broad or bold claims. Our scientific and technological development in various areas and at various times is recorded, and the impact of those developments is seen in context and as an indication of how our society has used and managed science for our benefit. A significant challenge for Curriculum for Excellence and the Science for Excellence series is to change our pupils' attitudes to science and to help them become more able to engage positively in issues that will affect them. It is intended that the series' approach and content will help them to appreciate the scientific challenges and issues facing mankind and to respond in critical and informed ways. Science for Excellence strives to act as a sound preparatory text for pupils, including those progressing to the next stage, providing a secure understanding of the key issues in science.
|Chambers, Paul ORCID: https://orcid.org/0000-0002-1133-3447 and Jeffrey, Stephen ORCID: https://orcid.org/0000-0002-8280-4250; Souter, Nicky
|Item type:
|ID code:
|education, teaching science, physics, physical science, science education, curriculum, Curriculum, Science (General)
|Education > Theory and practice of education > Curriculum
Science > Science (General)
|Faculty of Humanities and Social Sciences (HaSS) > School of Education > Education
|Depositing user:
|Pure Administrator
|Date deposited:
|18 Nov 2011 11:27
|Last modified:
|31 Jul 2020 00:23
|Related URLs:
|Export data:


text:
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text:
"Religious Diversity Must Make Its Mark in History Lessons"
On 22 November, German Federal President Frank-Walter Steinmeier hosted the award ceremony of the German history competition 2016/17 in Bellevue Palace in Berlin, his official residence. More than 5,000 students conducted local and family history projects on the topic of »God and the world. Religion makes history«.
The competition's results have been assessed by the German expert Frank-Michael Kuhlemann who holds the Chair of Modern and Contemporary History and Didactics of History at the Technical University of Dresden. In an interview with Bernd Vogenbeck, Program Manager at the Körber Foundation, Mr. Kuhlemann stresses the importance of religion for history lessons.
BV: Mr. Kuhlemann, were you surprised by the positive response to the competition topic »Religion«?
FMK: The participation figures show that the subject of religion is something that moves young people. However, apart from individual cases, this is not significantly related to belonging to a religious community. This is shown partly by the choice of topics of many young people, and it is also indicated by the distribution of the contributions: Both the federal states in northern Germany, which are characterised by a more liberal understanding of religion, and the federal states in eastern Germany where religious affiliation is low, participated very actively. I interpret this success in a different way: Many young people concern themselves with meaningful elements of religion and question the meaning of beliefs for their identity – this interest in the topic is reflected in the contributions.
BV: What do you think attracted young people to the topic?
FMK: It is particularly the moral problems that arise from personal religious beliefs and the political side of religion: What is right? And what is wrong? How can we assess the actions and activities of people and communities? Have people stood up for "good" or for ideals and have they offered resistance where necessary? These are value issues in which young people seek orientation and which involve a high level of identification and motivation.
BV: What conclusions can be drawn from this for standard history lessons?
FMK: The contributions make it clear that religion is seen as an important factor in history – not as a dogma, but as a value entity that is an integral part of social problems, is at the same time negotiable and calls for participation. The interest of the pupils reflects current socio-political issues and at the same time takes up scientific discourses. All these points justify treatment in history lessons, and yet religion is still faced with a difficult situation in this context: Its significance in contemporary history often remains unacknowledged because it is perceived as being suspicious, or it is marginalised as not playing a key role in shaping history. However, the significance and diversity of religious beliefs are part of our society – and this must also be reflected in history lessons!
BV: Where do you see points of contact for the topic of religion in history lessons?
FMK: The competition entries cover a broad spectrum – from the history of violence to art and architecture. I see implications for the traditional orientation of history lessons above all where connections between religion and politics can be established. However, approaching the history of everyday life and mentalities can also open up new perspectives in history lessons: A history of labour migration during industrialisation or the integration of displaced persons after 1945, taking into account customs and welfare, would allow completely different insights to traditional economic or social historical approaches. Questions of identity and integration that could be posed in this way are relevant to pupils for age-related reasons and important in order to reflect on the challenges of a pluralistic society.
BV: The topic "lslam" was only taken up in less than four percent of the competition entries – how would you interpret that?
FMK: The reasons for this are undoubtedly as varied as the contributions themselves. This figure shows that, despite the now sixty-year-old presence of Muslim communities in Germany, they have only been addressed as part of our society in recent years. The assertion by former Federal President Wulff that Islam belongs to Germany has not yet led to a perception that Islam has also become part of more recent German history. In addition, there is – besides questions regarding the availability of sources – some uncertainty among teachers and students when dealing with the topic.
BV: What role should religious diversity play in history lessons in the future?
FMK: Multi-culturalism and multi-ethnicity may have become part of the historical didactic debate, but the curricula still have a lot of catching up to do. Furthermore, religion in particular, which is what often accounts for this diversity, is hardly taken into consideration. The fact is that it can be understood not only as a factor of diversity, but also as an inclusion factor: Dialogue only becomes possible with the understanding of one's own values as well as the values and world view of others.
BV: Thank you!


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Thanksgiving Hostess Gifts
Can you believe that Thanksgiving is next week?! I KNOW!! But don't stress out too much, because I have got your back! Not only am I sharing some adorable hostess or guest gifts today from my fabulous Paper Crafting Contributor Eden- BUT I am also sharing even more helpful ideas this weekend! But first things first, let's hand it off to Eden!
Hello all! Eden here and I have some more great ideas using SVGs, my Cricut Explore and some of my other favorite tools! I have made 3 gifts today that work great for kids, guests and hostesses so let's get started! We will start with the hostess- what hostess doesn't
need want a bottle of wine? Exactly.
Wine Bottle Tag Materials:
- From Jaded Blossoms: Wine Tag Die, 1/2 of a Candy Wrap Die to form the tag, "For the Hostess" Stamp set
- From SVG Cutting Files: Pilgrim Boy svg from the file called Pilgrims (which also includes a little girl pilgrim)
- Other Materials used: Cuttlebug embossing folder called Thistle Tile by Anna Griffin. Twine and paper from my stash.
After finding the Pilgram SVG I started the process of cutting all the pieces and doing this can take a lot of time. In order to cut down as much time as possible when cutting I arrange the paper all on the same mat to cut everything at the same time.
Then everything got pieced all together!
Now let's move onto the Candy Holder- here is what I used, also great for a hostess:
- From Jaded Blossoms: Candy Caddy Die, Candy Wrap Dies, "For the Hostess" Stamp Set, "Thanksgiving Candies" Stamp set
- From SVG Cutting Files: Turkey svg was from a file called Gobble til you Wobble
- Other materials used: Paper & twine from my stash and the grass & clouds were cricut cuts
I arranged the paper in the same way to save time, just make sure the measurements match up with your screen.
Here is the finished product after it was pieced together and embellished!
Lastly, I have a much easier option, this would be great on the table or as a favor and Michelle may have been able to put together a printable for all of you who don't yet have the Cricut Explore as well!
For this one I designed it all in the Cricut Design Studio and used the Print and Cut feature to print it out then cut out the rectangle onto Cricut Sticker Paper.
Then I mounted that on orange cardstock and the 5 inch by 2.5 inch size is perfect for the standard Ziploc bags full of chocolate treats!
Thanks so much for having me!
They are all so cute! Thank you so much Eden for sharing these adorable gifts with everyone! As promised, if you don't have a Cricut Explore (like me!!! It is on my birthday list though!!) then I have a made a printable for you to use to put together the cute little Thanksgiving bags with the chocolates. I think I might have to make these for my daughter's class!
Here is the printable, just cut this out and mount it on cardstock and you are good to go!
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Ask a Health Coach
Q: My Heart Age is a little high. What's the best way to reduce it?
Your Heart Age, which is calculated as part of your annual HealthCheck, is an estimate of your cardiovascular health based on a number of inter-related factors, including your age, gender, and your specific HealthCheck measurements.
The key to reducing Heart Age is to make improvements in the risk factor(s) that are a problem for you. The five risk factors include:
- Blood Pressure
- Body Weight
- Tobacco
- Blood Sugar
- Cholesterol
Work with your health coach to understand your results and target specific improvements you can make to reduce your Heart Age. You might be surprised at the results. For example, if you smoke cigarettes, just by quitting you can reduce your Heart Age by four years.


text: "The Rider" is the first kid's bow tie I created. He is very fond of children with his happy animals. This boy's bow tie is made with a cotton fabric. It measures 9 cm wide and 4 cm high and fits all necklaces with an attachment system that is adjustable from 25.5 cm and up to 40 cm.


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Our party will include games and treats, including cupcakes for everyone who purchased tickets to decorate. We'd like to give you all of the ingredients ahead of time. All ingredients that will be used have been confirmed to be gluten-free by the manufacturer.
The cupcake frosting will be made at Starry Lane Bakery, a dedicated gluten-free bakery in Hillcrest. Their products are also free of all major allergens, including dairy. The frosting is made with:
The GF King Arthur cake mix (for those who can eat dairy) will also use:
The Pamela's cake mix (for those who can't eat dairy) will use:
The cupcakes will be prepared in the all-gluten-free household of one of our Board members.
We also plan to offer water, string cheese, and mini-clementine oranges.
Leave a Reply.
The Board of the National Celiac Association, San Diego
March 2020


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"Come in and go out radiant! That's the feeling you'll get every time you walk in to ColourWorld Beauty & Grooming, a new and upscale destination for all your beauty needs." Colourful World Beauty & Grooming
I went to wish Mr Ato DeGraft-Johnson a very happy birthday via Facebook and as always -checked out his photos. Truly loved and admired, Mr Ato DeGraft-Johnson's tagged by most high-end businesses and artists in Ghana. And one such business is Colourful World Beauty & Grooming located at: No. D7, 1st Labone Street, Accra, Ghana. Their number is: 0302958063. I can't find their website [?????] but you can check them out via their Facebook fan page at: http://www.facebook.com/add_email.php#!/pages/ColourWorld-Beauty-Grooming/116837615079339


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Doing Business in Stephens County
Welcome to Toccoa-Stephens County! Read our Welcome Letter in 6 different languages.
Since 2010, 8 industries have located or expanded in Stephens County, creating over 400 new jobs and adding more than $60 million in new private investment in the community. These companies have constructed or occupied facilities totaling more than 432,000 square feet on over 100 acres of newly developed or redeveloped land.
Our diverse business sector includes local as well as foreign investment. Four Stephens County companies have foreign ownership, and twenty-two companies have foreign branches and/or do business internationally.
Starting a Business in Stephens County
How can you become an entrepreneur? How can you start your own business? The Toccoa-Stephens County Chamber of Commerce and the Stephens County Development Authority have designed a booklet to simplify the transition into the role of an entrepreneur. Starting a Business in Stephens County will make establishing your own business easier by giving you "one-stop shopping" for the information you will need. We are determined to promote economic growth and development. By giving you the proper tools, we can help build a strong economic foundation.
For additional information, we provide the Six C's that Should be in Every Business Plan.
|Learn More About
Starting a Business in
Toccoa-Stephens County
|Learn More About
Starting a Business in
Downtown Toccoa
The SCDA is responsible for the recruitment of new businesses including industrial, manufacturing, distribution, corporate and regional headquarters and customer service centers. It also provides support and assistance with all other types of economic development projects.
The SCDA also works with existing businesses and industries to ensure their continued success. While recruitment of new industry is a significant function, aiding in existing industry expansion and retention is just as significant. About 80 percent of all new jobs in Georgia come as a result of existing industry expansion.
For BusinessesIncentives
Georgia Work Ready
State Tax Credits
Opportunity Zone Tax Credit
Freeport Tax Exemption
Training and Education
Grants & Other Finance Options
Drugs Don't Work
Latest News
Contact Us
- Phone:
- 706-886-4242
- Address:
- 31 W. Doyle Street, Toccoa GA 30577
- Hours:
- Mon-Fri 8:30 am to 5:30 pm
- E-mail:
- email@example.com
#Request.Business_City# #Request.Business_State# #Request.Business_Zipcode#


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Xmas Holidays from 25 to 26
The SMOK Spirals Replacement Coil Pack features an enlarged coil structure compared to the size of the chassis that integrates eight wicking ports for rapid and effective wicking. Each Spirals Sub-Ohm Tank includes a 0.6 ohm and 0.3-ohm Dual Core head, rated for 18 to 35W and 20 to 45W, respectively. SMOK has specifically designed the coil structure and chimney to ensure maximum flavour performance coupled with ample wicking ports to ensure consistency during extended use.
Coil Specifications:
Use spaces to separate tags. Use single quotes (') for phrases.


text:
Gottex Vista Red Square Neck One Piece Swimsuit
As low as $124.99 Regular Price $158.00
In stock
Clearance - All Sales Final
Item: 19VS173-600
A solid group with shirring to compliment the printed groups. Shirring in different directions flatter the body and accentuate the waistline.
Consult size chart for perfect fit.
- Soft cup bra with adjustable straps
- Square neck
- Solid color with shirring
- 59%Polyamide 41% Elastane Knitted


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Key considerations when dealing with space and translation
In the previous sections we have seen how translation can both use and transform space. These are some of the key considerations which have emerged from our discussion:
1: Type: what type of translation (or interpreting) is appropriate in a given scenario?
2: Visibility: how visible (or invisible) is the translation going to be in a specific place, and why?
3: Location: what are the physical locations in which translation will take place and how can they be adapted, if needed, to ensure that the space is suitable for the activities that are being planned?
4: Participants: who are the people taking part in the translation process and what is the relationship among them?
5: Power relationships: are there any power implications in the situation and, if so, how are they going to influence the translation process or its outcomes?
6: Ethical issues: what are the ethical questions posed by the specific situation in which translation will take place?
© Cardiff University


text: Family holidays in spring needn't mean soggy BBQs and missing tent pegs. England is packed with fun and engaging things to do with kids, from dizzying treetop adventures to interactive museum exhibits and thrilling theme park breaks. For more of a laid-back pace, escape to a Cotswolds farm stay where you can spot the beasties on a nature trail and befriend the farmyard's residents. How about a once-in-a-lifetime experience you'd never expect? Let the roar of tigers and lions lull you to sleep as you bed down on a bespoke luxury safari in the deepest, darkest wilds of... Kent.


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I've been a bad blogger and will continue to be for a couple more weeks because……the goat needs milking, the gardens need tending, the windows need washing and the pool could use a little cleaning. Just CAN'T seem to keep up with all the demands of summer. See why I truly ENJOY winter! Outside chores done by 4:30 and inside chores done by 6:00. Beats the hours we tend to keep this time of year. I'm not complaining just overwhelmed at times.
Thursday night our fresh air child arrives from inner city New York for a 2 week visit. This is her second year coming to the farm. We are all very excited for her return. In addition to that we will be hosting a pig roast for Maine Man & 2 of his friend's 40th birthday in the next couple weeks. I'm sure I'll have some good pictures to post of that event.
Additionally I'm still reading Bringing Food Home ~ The Maine Example by Merry Stetson. I'm loving that book and I've highlighted lots. Yesterday I received Flower Girl's home school curriculum in the mail….paid for it with rolled change but we managed. Now that I've looked through it the fear factor has set in. I also received a book that was sent to me so that I would read and do a book review, "The Back to Basics Handbook". I think it may be fall before that happens.
P.S Tonight the kids cooked me up some crayfish from the pond and served it w/ melted butter. I must admit it was not bad, tasted like lobster just much smaller. Love those country kids!


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So I've been looking into dethatching and aeration and has some questions that I couldn't find straightforward answers for during my searches. Figured I'd see if anyone had any thoughts on here. My lawn has never been dethatched or aerated, it's about 2 years old now so it is overdue for at least one these. My questions are whether I should do both in the same season? Or should I space out the two? It seems it would be best to space them out since both do damage the lawn initially. Also, if they do need to be spaced out which would be best to do first? Thanks!
Great question to raise. It's a tough one to answer because there doesn't appear to be a definitive answer. There is not a lot of evidence pointing to whether they should be done together, separately, or if that even matters.
Instead, it's best to focus on the type of grass you have, e.g. cool season or warm season grass. This way you can make sure if you go with one or the other, or both you are doing it at the right time of the year. Whichever you decide, doing it at the right time of the season will prevent damage to your yard and lead to a healthy looking lawn.
This is just a suggestion but I would start with aerating because the process will break up the thatch.
I hope this helps.
I've been wondering the same thing… I have zoysia grass in FL. Most articles I've read says the thatch layer should remain manageable under normal circumstances. The older thatch decomposes into the soil as fast as the newer clippings are added. If you over fertilize and under-mow. this might not be the case.
This article makes a good argument for aeration over thatching, as the plugs will help manage/decompose the thatch layer. Happy Reading!
If your core aerate I would not flail dethatch, it's a lot to put on the stolons. I would buy a thatching rake and hit the problem spots and aerate or vice versa.
I have done both of those in the same season on a few occasions right after each other.
A flail dethatch we will beat the hell out of your yard and stresses it big time
I personally core every other year and verticut every year. Verticutting thickens the grass most impressively. If you can't find a verticutter to rent, search for a slit seeder.
OK yeah I think I've settled on core aerating this weekend and de thatching next year. Thanks
Just curious, how did you get your hands on a verticutter? I see those all the time on the golf course but not so much in residential. Will have to look into it more. Not sure if they make a push version but my lawn has all kinds of curves and tight spot so a tractor pulled version would miss a bunch. Will have the same problem with an aerator…
Good suggestions tho… thx.
Look for a slit seeder. For residential purposes it's the same thing
Blue bird makes a real pos that bought for 300
ha, so don't buy the blue bird?? Looking to see if Home Depot or John Deere has rentals…
I don't regret buying it but it is what it is
Is an over-seeder the same thing as a slit seeder? Home Depot has a Classen TS-20 turf overseeder for rent.
i think that will do, but set it deep,
Hi Johnny,
Zoysiagrass is famous for developing a thick layer of thatch, which becomes a breeding grounder for turf diseases. Check out turf management publications from the University of Florida. Now would be a great time for core aeration in Florida. But I don't want to guess on the month to detach your turf. Maybe January? I attended the Oklahoma turf conference last December and a professor from Arkansas showed a photograph of Zoysiagrass with 7 inches of thatch!
Jesus, at that point, kill it with fire!
Thanks for the info @robertkc. My zoysia was just installed a year ago with new construction so I don't think I've built up a 7 inch thatch later just yet
Looking into an aerator now because one side of the lawn was heavily compacted when they dug out the pool. The grass there is only about 0.5-1 in high and the roots aren't that deep… <2 inches, which is pretty much still just the sod. Hoping some aeration gives it room to breathe
Sounds good. Do you have a Flatwoods soil - Sandy on top with a clay pan beneath?
Didn't know that was the term for it but that's exactly what I have. I'm told it's a combination of non-naive topsoil and clay dug out from man made lakes they used to build up the development.
That is it exactly.


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Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life. Particularly, the diverse structures of the outermost boundary layers strongly reflect adaptations of organisms to specific ecological and environmental conditions (6).
Over the past 3 decades of research, it has become apparent that one of the most common surface structures on archaea and bacteria are monomolecular crystalline arrays of proteinaceous subunits termed surface layers or S-layers (125, 131, 132). Since S-layer-carrying organisms are ubiquitous in the biosphere and because S-layers represent one of the most abundant cellular proteins, it is now obvious that these metabolically expensive products must provide the organisms with an advantage of selection in very different habitats (133). This minireview provides a brief survey of the current state of our knowledge about S-layers with a particular focus on molecular biological and genetic aspects. Other recent reviews (5, 7, 127, 133, 135) are recommended for a more detailed introduction to and treatises on this subject.
S-layers have now been identified in hundreds of different species belonging to all major phylogenetic groups of bacteria, and they represent a feature common to almost all archaea (for a recent compilation, see reference 133). This widespread occurrence on prokaryotic organisms has not always been appreciated, since S-layers are often lost during prolonged cultivation under laboratory conditions. Consequently, fresh isolates should be examined by electron microscopic techniques as soon as possible, preferably by freeze-etching of pellets of unwashed cells (Fig.1) in complete medium (64, 125). For high-resolution studies on the mass distribution of S-layer lattices, negatively stained preparations of isolated or recrystallized S-layers have been used (for reviews, see references4, 5, 50, and 130). More recently, high-resolution images of S-layers were also obtained by applying underwater atomic-force microscopy (38, 135).
The S-layer lattices can have oblique (p1, p2) square (p4), or hexagonal (p3, p6) symmetry. The data now available show that hexagonal symmetry is predominant among archaea (for a compilation, see reference133). Depending on the lattice type, one morphological unit consists of one, two, four, three, or six identical (glyco)protein subunits, respectively, and they exhibit center-to-center spacings of approximately 2.5 to 35 nm. Most S-layers are 5 to 25 nm thick, and they reveal a rather smooth outer surface and a more corrugated inner surface. Among S-layer lattices of archaea, pillar-like extensions on the inner surface can be observed (80, 99, 100, 103). Since S-layers are monomolecular assemblies of identical subunits, they exhibit pores identical in size and morphology. In numerous S-layer lattices, two or even more distinct classes of pores (generally in the 2- to 8-nm range) could be identified, occupying up to 70% of the surface area. Since S-layers are found in gram-positive and gram-negative bacteria and archaea, they can be associated with quite different supramolecular cell envelope structures. In gram-positive bacteria and archaea, the S-layer subunits are linked to the peptidoglycan-containing layer or to the pseudomurein. In gram-negative bacteria, attachment involves components of the outer membrane (e.g., lipopolysaccharides [LPS]). In archaea lacking a rigid wall layer, S-layers are the only wall component, being closely associated with the plasma membrane (38, 59).
S-layers represent unique model systems for studying the dynamic process of assembly of a supramolecular structure during cell growth and cell division. Different methods have been developed for the detachment of S-layers and for their disintegration into protomeric units (5, 62, 90, 133). Most S-layer proteins can be solubilized with high concentrations of agents that break hydrogen bonds (e.g., guanidine hydrochloride). Particularly S-layer proteins from gram-negative bacteria may be disintegrated by applying metal-chelating agents or cation substitution. These data have shown that the individual subunits of S-layers interact with each other and with the supporting cell envelope components through noncovalent forces. Isolated S-layer subunits frequently maintain the ability to recrystallize into regular arrays in suspension or on surfaces (including that cell envelope component they were originally associated with) upon removal of the agent used for their isolation (5, 105, 115, 126, 133). Up to now, the most detailed in vivo and in vitro assembly studies were performed with S-layers from members of the family Bacillaceae (115, 129, 133). It was shown that differences in the surface net charge and in the hydrophobicity of the inner and outer surfaces, binding domains specific to components of the supporting rigid envelope layer and defined intersubunit binding properties, are responsible for the proper orientation and incorporation of the S-layer subunits on cell surfaces. From a more general point of view, it is now evident that S-layers are dynamic closed surface crystals with the intrinsic ability to continuously assume a structure of low free energy during cell growth and division. In most bacteria and archaea, the rate of synthesis of S-layer subunits appears to be strictly controlled, as at different growth rates only small amounts are detectable in the medium. Analysis of the lattice faults in S-layers of lobed archaea possessing hexagonal arrays as the exclusive wall component provided strong evidence that complementary pairs of pentagons and heptagons play an important role as sites for the incorporation of new subunits in the formation and maintenance of the cell structure and in the fission process. The latter was assumed to be dependent on the ratio of the increase in protoplast volume to the increase in actual S-layer surface area during cell growth (106).
Chemical analyses of isolated S-layers showed that they are mostly composed of a single protein or glycoprotein species with apparent relative molecular weights of 40,000 to 200,000. Only the S-layers of a few organisms, such as that of Clostridium difficile(140) and Bacillus anthracis (39, 84, 85), consist of two types of S-layer subunits which together form a defined lattice type but do not cross-react with polyclonal antibodies. S-layer protein EA1 of B. anthracis was proposed to establish the main lattice, whereas the second S-layer protein, Sap, is most probably only incorporated into this template. Two superimposed S-layer lattices consisting of different types of S-layer subunits were identified in the cell envelopes of Brevibacillus brevis(formerly Bacillus brevis; 154) andAquaspirillum serpens (58).
Amino acid analyses revealed that S-layer proteins of organisms from different phylogenetic branches are rather similar in overall composition (5, 115, 128, 133). Typically, S-layer proteins possess a high content of acidic and hydrophobic amino acids. Lysine is the predominant basic amino acid, while the arginine, histidine, and methionine content is generally low and cysteine was only detected in a few S-layer proteins (86, 115). According to sequence analyses, the isoelectric points (pIs) of most S-layer proteins are in the weakly acidic pH range. An exception was found for the S-layer proteins of Methanothermus fervidus (17) and for different lactobacilli (11), which possess pIs of 8.4 and 9 to 11, respectively. According to circular-dichroism measurements, approximately 20% of the amino acids are organized as α helices and about 40% occur as β sheets. Aperiodic foldings and β-turn content may vary between 5 and 45%. Secondary-structure predictions based on protein sequence data (see Table 1) revealed that most α-helical segments are arranged in the N-terminal part of the proteins. For the remaining part of the sequences, mainly short β-strands connected by loops and turns have been predicted.
S-layer proteins of many archaea and those of gram-positive bacteria can possess covalently bound carbohydrate chains (for reviews, see references 70, 86, 87, and 89). Typically, the glycan chains are composed of 20 to 50 identical repeating units containing neutral hexoses, pentoses, heptoses, 6-deoxyhexoses, and amino sugars. The glycan chains of S-layer proteins from gram-positive bacteria are more similar to the LPS occurring in the outer membranes of gram-negative bacteria than to eukaryotic glycan structures (118). Core oligosaccharides of S-layer glycoproteins consist of two to four sugar residues attaching the carbohydrate chains mostly via O-glycosidic linkages (galactose–tyrosine, glucose–tyrosine,N-acetylgalactosamine–serine, N-acetyl galactosamine–threonine) to the protein moiety (86, 87, 153). However, in a few examples, N-glycosidic linkages (asparagine–rhamnose) have been observed (86, 87). S-layer glycoproteins of gram-positive bacteria possess two to four glycosylation sites, whereas up to 25 glycosylation sites have been determined for those of archaea in which N-glycosidic linkages predominate (70). The presence of sulfate residues in the glycan chains of the extremely halophilic bacterium Halobacterium halobium (82) endows the S-layer lattice with a strong net negative charge. Since only neutral glycan chains were identified for the S-layer protein of the moderate halophile Haloferax volcanii (81), it was suggested that the negative charges are required for stabilizing of the S-layer lattice (138). A different type of posttranslational modification than glycosylation was reported for the S-layer protein ofAeromonas hydrophila. Phosphorylation of tyrosine residues decreases the pI of this S-layer protein from 6.7 to 4.6 (141).
During the last decade, numerous S-layer genes from organisms having quite different taxonomical positions have been cloned and sequenced (for reviews, see references 66 and135). A survey of all of the sequenced S-layer genes is given in Table 1. Although it was proposed for several years that sequence identities among S-layer proteins are extremely rare or do not even exist, it is now evident that high sequence identities are limited to the N-terminal region, which is responsible for binding of the S-layer subunits to the underlying cell envelope layer (135). On the other hand, only low sequence identities (∼20%) were found for the middle and C-terminal parts, comprising those domains that are involved in the self-assembly process and that are exposed inside the pores and on the S-layer surface. Despite these low sequence identities, conserved four- to six-amino-acid sequences were observed at relatively constant distances over the whole middle and the corresponding C-terminal parts of S-layer proteins from different Bacillus spp. (SbsC, Sap, CTC, OlpA, and SbsB; Table 1), most probably assembling into oblique lattices (53). Considering the highly competitive situation of closely related organisms in their natural habitats, it is obvious that the S-layer surface has to contribute to diversification rather than to conservation. With respect to this, the importance of S-layer variation leading to the expression of alternative S-layer genes under different stress factors such as those imposed by the immune system of a host in response to an S-layered pathogen or drastic changes in the growth and environmental conditions for nonpathogens is conceivable (29, 112).
For S-layered bacteria with a generation time of 20 min, approximately 500 S-layer subunits have to be synthesized per second, translocated through the cell wall, and incorporated into the existing S-layer lattice (128). To keep the bacterial surface completely covered with a closed protein lattice, promoters preceding S-layer genes must be very strong (66). Actually, the promoter of the S-layer gene of Lactobacillus acidophilus is twice as efficient as that preceding the gene encoding lactate dehydrogenase (12, 13, 104), which is considered to be one of the strongest promoters in bacteria. Two promoters were identified for the S-layer gene of L. brevis ATCC 8287 (55),A. salmonicida (24), and B. stearothermophilus ATCC 12980 (53), which are most probably used during different growth stages. The P2 promoter preceding the S-layer gene sbsC in B. stearothermophilusATCC 12980 seems to be 2.4 times more frequently used in exponentially growing cells than the P1 promoter (53). The existence of three tandemly arranged promoters has been reported for thecwp operon of B. brevis 47 (1), an organism which concomitantly produces two different types of S-layer proteins that assemble sequentially into superimposed S-layer lattices. The promoter of the slpA gene encoding the S-layer protein of L. brevis (147) was recently exploited for heterologous expression of proteins and was recognized inLactococcus lactis, L. plantarum, and L. gasseri (54).
With the exception of the S-layer proteins of the gram-negative bacteria Caulobacter sp. and Campylobacter sp., all of those sequenced to date are produced with an N-terminal secretion signal peptide which is cleaved off after translocation through the plasma membrane. Linker peptide insertions at the C-terminal end of the S-layer protein of Caulobacter crescentus (Table 1) indicated the existence of a type I secretion signal (8, 9). A similar observation was reported for the S-layer protein of Campylobacter fetus, which also reveals potential secretion signals at the C-terminal part (142). InSerratia marcescens, the slaA gene is located in the upstream region of the lip exporter genes and most probably encodes an S-layer protein (56). The putative S-layer protein is strictly recognized by the lip system, which belongs to the ATP-binding cassette exporter and which is responsible for signal peptide-independent secretion of a lipase and a metalloprotease. On the other hand, the S-layer proteins VapA and AhsA of the gram-negative bacteria A. salmonicida and A. hydrophila are produced with either a 21- or a 19-amino-acid-long signal peptide (23, 24, 141). A conserved gene (abcA) which is located immediately downstream of thevapA gene and affects its expression in Escherichia coli was identified. The corresponding AbcA protein possesses P-loop-containing residues at the N terminus, suggesting that this protein has ATP-binding activity and belongs to the ABC family of transport proteins (25). Further, it could be demonstrated that AbcA is a bifunctional protein in which the N-terminal part regulates the synthesis of the O-polysaccharide side chains of the LPS, functioning as a binding site for the VapA protein. The C-terminal leucine zipper increases expression of the vapA gene and can thus be considered as a transcriptional activator of the P2 promoter (25, 95, 96).
The existence of a translational autoregulation system was reported for the S-layer protein SlpA of Thermus thermophilus HB8 (41). Three genes which specifically repressed the expression of the S-layer protein promoter were cloned in E. coli. Sequence comparison revealed that one of these genes encodes a protein (Rep54) which corresponds to the C-terminal fragment of the S-layer protein and could bind to the 5′-untranslated region of the S-layer protein mRNA.
In gram-positive bacteria, the rigid cell envelope layer is composed of peptidoglycan and accessory (secondary) cell wall polymers which may be teichoic acids, teichuronic acids, lipoteichoic acids, or lipoglycans (2, 93). The polymer chains are either covalently linked to the peptidoglycan backbone via phosphodiester bonds or tethered to a lipid anchor moiety (2). Most of the biological functions discussed for secondary cell wall polymers, such as binding of cations, keeping of the peptidoglycan sacculus in an expanded state by charge repulsion, binding of protons to create an acidic cell wall during bacterial growth and division, or provision of a physical barrier to prevent diffusion of nutrients and metabolites, were seen in context with their net negative charge, and only forB. subtilis were the teichoic acids reported to serve as a binding site for a cell wall autolysin (49).
By sequence comparison, S-layer-homologous (SLH) motifs (74) have been identified at the N-terminal part of many S-layer proteins (14, 26, 33, 39, 40, 51, 65, 71, 72, 85, 97, 144) and at the C-terminal end of cell-associated exoenzymes (71, 78, 79) and other exoproteins (71, 73) of gram-positive bacteria. According to their origin (S-layer proteins, cell-associated exoproteins, porins), SLH motifs have been divided into three main groups whose specific properties have recently been reviewed by Engelhardt and Peters (38). Typically, S-layer proteins and cell-associated exoenzymes possess three repeats of SLH motifs each consisting of 50 to 60 amino acids. Due to their wide distribution among gram-positive bacteria, SLH motifs were suggested to anchor cell-associated exoproteins permanently or transiently to the cell surface (74). In contrast to the original assumption that peptidoglycan functions as a binding site (74), it is now evident that secondary cell wall polymers serve as anchoring structures for many S-layer proteins, cell-associated exoenzymes, and exoproteins (15, 22, 51, 72, 73, 83, 107).
First studies regarding the importance of secondary cell wall polymers for anchoring of S-layer proteins to the rigid cell wall layer (107) were carried out with the S-layer protein SbsB (65) from B. stearothermophilus PV72/p2, an oxygen-induced variant strain of B. stearothermophilusPV72/p6 (112). The S-layer protein SbsB carries three typical SLH motifs at the N-terminal part. Affinity studies using the whole S-layer protein (107) and an N-terminally truncated form (Δ3 SLH motifs; amino acids 208 to 920; Moll, unpublished data), as well as native peptidoglycan-containing sacculi and those extracted with hydrofluoric acid, leading to pure peptidoglycan, revealed that the N-terminal part recognizes a secondary cell wall polymer composed mainly of N-acetylglucosamine andN-acetylmannosamine and not the peptidoglycan as a binding site (107). However, affinity studies with proteolytic cleavage fragments indicated the coexistence of a binding region for peptidoglycan and the secondary cell wall polymer on the N-terminal part of this S-layer protein (111). The isolated secondary cell wall polymer had unique effects on the S-layer protein SbsB (110). Firstly, the presence of the secondary cell wall polymer inhibited the in vitro self-assembly of the guanidine hydrochloride-extracted S-layer protein and kept the S-layer protein in a water-soluble state. Under these conditions, the S-layer protein showed a significantly enhanced tendency to recrystallize into closed monolayers on those solid supports to which the subunits bind with their outer surface. Secondly, the polymer chains protected the S-layer protein against proteolytic attack and specifically masked an N-terminal endoproteinase Glu-C cleavage site. Preliminary studies indicate that a highly specific lectin-type recognition mechanism (110) exists between the S-layer protein and this type of secondary cell wall polymer.
More recently, the complete structure of the secondary cell wall polymer functioning as a binding site for the SLH motifs of the S-layer protein from B. sphaericus CCM 2177 was provided by nuclear magnetic resonance analysis (51). This secondary cell wall polymer is a teichuronic acid and is composed of disaccharide repeating units having the structure →3)-[4,6-O-(1-carboxyethylidene)]∼0.5-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→. In contrast to the SLH motifs of most S-layer proteins, which reveal a positive net charge, those of B. sphaericus strains (14, 26) are net negatively charged, which explains why bivalent cations are required for binding of the S-layer subunits to the rigid cell envelope layer (51).
Experimental evidence that the SLH motifs recognize a cell envelope component that could be extracted from peptidoglycan-containing sacculi with hydrofluoric acid was further provided for the S-layer proteins Sap and EA1 of B. anthracis (22, 85) and SlpA ofC. thermocellum (22, 72) and the S-layer protein and cell wall-associated xylanase of Thermoanaerobacterium thermosulfurigenes EM1 (15). In affinity studies in which peptidoglycan-containing sacculi were used as a binding matrix, the Kd values for the polypeptides comprising either the SLH motifs of EA1 and Sap were 1.8 × 10−7and 1.0 × 10−7 M, respectively (22). Similar values were determined for the SLH motifs and peptidoglycan-containing sacculi of C. thermocellum(22). In all cases, pure peptidoglycan was unable to bind the SLH motifs (22).
In contrast to most S-layer proteins of gram-positive bacteria, those of B. stearothermophilus wild-type strains (53, 65) and Lactobacillus (11, 12, 18, 92, 147) do not possess SLH motifs. Nevertheless, the N-terminal part of the S-layer proteins of B. stearothermophilus wild-type strains is highly conserved and recognizes a net negatively charged secondary cell wall polymer as the binding site (35, 53). This type of secondary cell wall polymer is composed of tetrasaccharide repeating units that contain glucose, N-acetylglucosamine, and 2,3-diacetamido-2,3-dideoxymannuronic acid in a molar ratio of 1 to 1 to 2 (117). Sequence analysis of the S-layer proteins fromB. stearothermophilus PV72/p6 and ATCC 12980 (Table 1) revealed an accumulation of arginine, lysine, and tyrosine at the N-terminal part. Because of a high density of positively charged amino acids, the N terminus possesses a positive net charge, which is in contrast to that of the whole S-layer proteins (53). Thus, bivalent cations were not required for binding of the S-layer subunits to the rigid cell wall layer (35), indicating the existence of direct electrostatic interactions between the N-terminal region and the secondary cell wall polymer. The accumulation of tyrosine in the N-terminal part is especially interesting, since in carbohydrate-binding proteins such as lectins, tyrosine interacts with N-acetylated sugars via hydrogen bonds (108, 152). The production of truncated forms of SbsC confirmed that the N-terminal part is not involved in the self-assembly process (35, 53) and seems to fold independently of the remainder of the protein sequence. According to secondary-structure prediction, about 70% of the N-terminal amino acids are organized as α helices.
As described above, the S-layer proteins of Lactobacillus do not possess SLH motifs (11, 12, 18, 92, 147). In earlier studies, Masuda and Kawata (77) reported that these S-layer proteins recognized a neutral polysaccharide but not teichoic acids or the peptidoglycan as a binding site. To conclude, the S-layer proteins from most gram-positive bacteria can be considered cell surface-located carbohydrate-binding proteins with the ability to self-assemble into monomolecular crystalline arrays (110). For a better understanding of the importance of secondary cell wall polymers for S-layer protein folding, prevention of self-assembly within the peptidoglycan-containing layer (16), and anchoring of the S-layer subunits to the cell surface, more detailed studies are required. According to the suggestion that S-layers may be considered cell surface-located carbohydrate-binding proteins, the N-terminal region of the S-layer protein of the gram-negative bacterium C. crescentus recognizes distinct oligosaccharides of the LPS as binding sites (9, 148). Two serotypes that depend on the type of LPS present in the outer membrane are distinguished in C. fetus, a pathogen for humans and ungulates (29). In type A cells, the S-layer protein SapA and its homologs are bound via a conserved N-terminal region of 184 amino acids to the type A LPS. The S-layer protein SapB and its homologs possess a different N-terminal region and recognize cells with type B LPS as a binding site (31, 32).
The example of Corynebacterium glutamicum ATCC 17965 demonstrated that in gram-positive bacteria, interactions other than that of the S-layer protein–polysaccharide type may exist. The S-layer protein of C. glutamicum ATCC 17965 has a hydrophobic C-terminal end which binds to a hydrophobic cell wall layer possessing a high content of mycolic acids (21). In archaea missing a rigid cell wall layer, the S-layer subunits closely interact with the plasma membrane. The S-layer protein of H. halobium shows a hydrophobic stretch of 21 amino acids on its C-terminal part which integrates into the plasma membrane (69). A similar observation was reported for the S-layer protein ofStaphylothermus marinus, which carries an extremely stable protease on its 70-nm-long stalk (80, 99).
The importance of insertion sequence (IS) elements for the generation of S-layer-deficient phenotypes has been demonstrated for at least three different organisms. In general terms, IS elements are small translocating DNA segments which occur on the host chromosome or on plasmids with the potential to move within and between bacterial genomes (76). Because of the existence of IS target sequences, transposition does not occur randomly. The integration of IS elements can either repress or activate genes located downstream. Variants of A. salmonicida, a fish pathogen, with S-layer-deficient phenotypes were generated by insertion of two different IS elements into different sites of the vapA gene and its promoter region (47). The bacteria with S-layer-deficient phenotypes were isolated after a temperature upshift from 20 to 30°C. While ISAS1 was unique among reported IS elements, ISAS2 showed strong similarities to transposases encoded by the IS30 family (76). However, both IS elements were found to be restricted to A. salmonicida, where they occurred in a low copy number. The insertion of IS elements was linked to the attenuation or complete loss of virulence (47). Temperature-dependent transposition was also described for IS4712 (120), an IS element which inhibits the expression of the S-layer gene sbsA (67) inB. stearothermophilus PV72/p6, leading to the S-layer-deficient phenotype strain PV72/T5 (112). Integration of IS4712 into the upstream region of thesbsA gene was induced by cultivating the organism at 67°C instead of 57°C. An S-layer-deficient phenotype variant of B. stearothermophilus ATCC 12980 was isolated from agar plates which were stored for at least 2 years at 4°C. PCR analysis revealed that the coding region of the sbsC gene was interrupted by a new type of IS element designated ISBst12, which currently cannot be attributed to any of the known IS families. Southern hybridization showed that ISBst12 was present in multiple copies in the S-layer-carrying strain, as well as in that with the S-layer-deficient phenotype (34). Interestingly, ISBst12 was also detected in B. stearothermophilus PV72/p6, its oxygen-induced strain variant PV72/p2, and the strain with the S-layer-deficient phenotype (PV72/T5). These findings are in agreement with the observation that organisms carrying one type of IS element tend to possess other types since multiple ISs can be delivered by a single vector (76).
S-layer variation leads to the expression of different types of S-layer genes or to the recombination of partial coding sequences. S-layer variation was studied in detail for C. fetus, an important pathogen for humans and ungulates (29), but was also observed for nonpathogens such as B. stearothermophilus(112, 121). In the case of pathogens, S-layer variation can be considered a kind of antigenic variation responding to the lytic activity of the immune system of the host cells. Both type A and B cells of C. fetus can produce multiple S-layer proteins with a single form predominating (31, 32). The apparent molecular weights of SapA and its homologs lie between 97,000 and 149,000, and depending on the molecular size, the S-layer subunits assemble into lattices with either hexagonal or square symmetry (42). Eight or nine S-layer gene cassettes are present in C. fetuswild-type cells and are tightly clustered on the genome in a region of less than 93 kb. Several studies confirmed that antigenic variation is due to the recombination of partial coding sequences (30, 31). In addition to promotor inversion between two oppositely oriented gene cassettes, one of the S-layer gene cassettes bracketing the invertible element with a nonflanking, previously silent gene cassette is exchanged. Thus, C. fetus uses a single promoter strictly by a single DNA inversion event at frequencies independent of DNA fragment size, permitting expression of different S-layer gene cassettes (29, 30). Although the S-layer protein SapB and its homologs possess an N-terminal region different from that of SapA, noncoding regions 300 bp upstream and 1,000 bp downstream of thesapA and sapB open reading frames, including promotor and transcriptional terminator sequences, were essentially identical for both types of cells (31).
Environmental factors inducing change in S-layer gene expression in nonpathogens were studied for B. stearothermophilus(112, 121). Under oxygen-limited growth conditions, thesbsA gene (67) encoding the S-layer protein SbsA, which assembles into a hexagonally ordered lattice type, was stably expressed during continuous cultivation of B. stearothermophilus PV72/p6 (112). After a change to non-oxygen-limited growth conditions (oxygen stress), expression of the second S-layer gene sbsB (65) was induced. The S-layer protein SbsB assembles into an oblique lattice and shows only 25% identity to SbsA. Change in S-layer gene expression was highly coordinated with the synthesis of a different type of secondary cell wall polymer (112). Interestingly, the S-layer protein SbsA recognized both types of secondary cell wall polymers as binding sites, guaranteeing complete coverage of the cell surface during the oxygen-induced switch. On the other hand, the S-layer protein SbsB showed only affinity for binding of the newly synthesized secondary cell wall polymer. Preliminary studies indicate that thesbsB gene is generated from partial coding sequences, while the sbsA gene is disrupted during the switch (121).
Although considerable knowledge has accumulated on the structure, assembly, biochemistry, and genetics of S-layers, relatively little information is available about their specific functions (5, 109, 127). Considering that S-layer-carrying organisms are ubiquitous in the biosphere, the supramolecular concept of a closed, isoporous, crystalline surface layer could have the potential to fulfill a broad spectrum of functions. In functional terms, S-layers must not be seen as isolated components since they are generally only part of more complex envelope structures.
Because S-layer lattices possess pores identical in size and morphology in the 2- to 8-nm range, they work as precise molecular sieves, providing sharp cutoff levels for the bacterial cells (113). The largest pores in the 5- to 8-nm range were found in S-layer lattices of those archaea lacking a rigid cell wall layer in which these crystalline arrays fulfill a shape-determining or shape-maintaining function (3, 4, 50, 80, 99). As a general feature, the S-layer subunits of archaea lacking a rigid cell wall layer possess long, hydrophobic protrusions which integrate into the plasma membrane. Thereby, a kind of periplasmic space is formed between the S-layer and the plasma membrane where secreted macromolecules involved in nutrient degradation, nutrient transport, and folding and export of proteins could be stored (46, 103). According to the proposed function, the S-layer glycoprotein of S. marinus is held at a distance of as much as 70 nm from the plasma membrane by membrane-anchored stalks. Furthermore, two copies of a hyperthermostable protease showing significant similarity to subtilisin are attached near the middle of each stalk. This enzyme is resistant to heat inactivation to 95°C in the free form and to 125°C in the stalk-bound form (80, 99).
S-layers from Bacillaceae were found to function as adhesion sites for cell-associated exoenzymes. The high-molecular-weight exoamylase from two B. stearothermophilus wild-type strains were bound to the S-layer surface in a density that did not disturb diffusion of nutrients or metabolites through the S-layer lattice (36, 37). S-layer-associated exoenzymes were also described for T. thermohydrosulfurigenes (71, 78, 79).
Regarding protective functions, S-layers from gram-negative bacteria such as A. salmonicida, C. fetus, A. serpens, and C. crescentus were found to protect the cells from attack by bacterial parasites such as Bdellovibrio bacteriovorus, but they could not shield them from other predators like protozoa (63). A quite interesting type of protective function was reported for the S-layer lattice ofSynechococcus GL-24 (122, 123), a cyanobacterium capable of growing in lakes with exceptionally high calcium and sulfate ion concentrations. The hexagonally ordered S-layer lattice functions as a template for fine-grain mineralization and is continuously shed from the cell surface to prevent clogging of further cell envelope layers.
S-layers can contribute to virulence when they are present as a structural component of the cell envelope of pathogens. The A-layer ofA. salmonicida endows this organism with high or intermediate resistance to the bactericidal activity of complement in immune and nonimmune sera. Furthermore, the A-layer plays an important role in uptake of porphyrins and shows unique immunoglobulin- and extracellular matrix protein-binding capabilities (28, 43). A similar observation was reported for the S-layer from B. cereus isolated from periodontal infections (61). Only the S-layer-carrying cells adhered to matrix proteins and were resistant to polymorphonuclear leukocytes in the absence of opsonins. The serum resistance of C. fetus was found to be due to the inability of complement component C3b to bind to the S-layer surface. Thus, effective opsonization occurred only after the addition of specific antibodies (29). In Rickettsia prowazekii and R. typhii, causing either epidemic or endemic typhus, the S-layer protein is responsible for humoral and cell-mediated immunity (19). In L. acidophilus, the S-layer is responsible for the adhesion of the bacterial cells to the intestinal epithelium (119).
During the last 10 years, studies on the structure, morphogenesis, genetics, and function of S-layers revealed that these isoporous monomolecular arrays have a considerable application potential in biotechnology, molecular nanotechnology, and biomimetics (for reviews, see references 105, 116, 134, and137). Many applications depend on the ability of isolated S-layer proteins to assemble into regular arrays in suspension or on suitable surfaces (e.g., silicon wafers, metals, and polymers) or interfaces (e.g., lipid films and liposomes) upon removal of the disrupting agent used for their isolation.
Since S-layer lattices are assemblies of identical (glyco)protein species, these crystalline arrays exhibit repetitive physicochemical properties down to the subnanometer scale and possess pores identical in size and morphology. S-layers from various Bacillaceaewere shown to be suitable for the production of isoporous ultrafiltration membranes with well-defined molecular weight cutoffs (113, 114, 134, 136). The S-layer lattice and the pore areas of S-layers contain functional groups (carboxylic acid, amine, and hydroxyl groups) which are aligned in well-defined positions and orientations. Accordingly, highly reproducible chemical modifications can be applied for optimization of molecular sieving properties and nonspecific adsorption (antifouling) characteristics (150, 151). The repetitive features of S-layers have led to their use as immobilization matrices for binding of monolayers of functional molecules (e.g., enzymes, antibodies, and immunogens) in a geometrically well-defined way. This application potential has been exploited for the production of bioanalytical sensors, immunoassays, affinity microparticles, and affinity membranes (116, 137).
Another line of development is directed toward the use of S-layer self-assembly products in suspension as a combined carrier-adjuvant system against infection with pathogenic bacteria in the immunotherapy of cancers and in antiallergic immunotherapy (52, 91, 135). On the other hand, S-layers of pathogenic organisms were identified to be essential for virulence. Whole-cell preparations or partially purified cell products are currently used as attenuated vaccines against fish pathogens (57, 143). Another broad application for S-layers is based on the ability of subunits to recrystallize into coherent lattices on functional lipid membranes (105, 135), including liposomes (68, 75, 94). These S-layer-stabilized lipid membranes mimic the supramolecular structure of those archaeal envelopes which possess S-layers as exclusive wall components or virus envelopes. They can be used in diagnostics, as vaccines, for drug targeting or delivery, and for gene therapy (115, 116, 135, 137). Alternative or complementary to existing S-layer technologies, genetic approaches are used to incorporate functional domains (proteins and glycans) in S-layer subunits while maintaining their ability to self-assemble into regular arrays (137).
Finally, S-layer lattices recrystallized on solid supports (e.g., silicon wafers) can be patterned by microlithographic procedures as required for lab-on-a-chip technologies (105, 135). The observation that S-layers of cyanobacteria may induce precipitation of minerals (122, 123) led to the concept of their use as templates for the formation of regular arrays of metal clusters as required in molecular electronics and nonlinear optics (27, 105, 124).
It is now evident that S-layers are the most common cell surface components of prokaryotic organisms. In comparison with the considerable accumulation of data concerning the structure, chemistry, assembly, and genetics of S-layer proteins, relatively little is known about their evolutionary relationship; their biosynthesis, including glycosylation; their transfer through intermediate envelope layers to sites of lattice extension; and their functional potential. Since S-layers of gram-positive and gram-negative bacteria can be lost in the course of subculturing in the laboratory, elucidation of their functional significance for a particular organism requires ecological approaches mimicking complex natural habitats and potential hazards. Although structurally diverse S-layers with barely any sequence identities in the constituent subunits are observed even among strains of the same species, these proteins must have domains with common structural and functional significance. In this context, it is important to remember that some organisms possess multiple S-layer genes and can even assemble different superimposed S-layers on their surface. This diversity and differentiation potential is further stressed by the observation that in S-layer proteins of strains of the same species can be either glycosylated or not (87, 88).
Finally, since S-layers can be considered supramolecular structures with the intrinsic ability to assume closed structures with low free energy during cell growth, it is tempting to speculate that S-layers, like membranes, could have fulfilled barrier and supporting functions as required by self-reproducing systems during the early periods of biological evolution (132).
This work was supported in part by the Austrian Science Foundation (project 12938) and by the Ministry of Research and Transportation.
We thank Dieter Moll for critical reading of the manuscript.
- Copyright © 2000 American Society for Microbiology
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