text:
The Bauhinia Foundation Research Centre shares research reports, occasional papers and weekly analyses on topical matters and current affairs in a timely manner. Want to stay connected with us? Please enter your email address.
(16 November 2012) The Bauhinia Foundation Research Centre today submitted a policy paper to the Financial Secretary's Office, advocating short and medium-term measures to relieve the burdens of the elderly, first-time home purchasers and regular long distance travellers.
The policy paper has been put together on the basis of the Centre's relevant research findings and taskforce discussions.
The Centre's Chairman Dr Donald Li said, "We appreciate the recent initiatives of the Government to promote the socioeconomic well-being of Hong Kong. However, the weakening momentum of global economic growth and widespread monetary easing worldwide have blurred the outlook of the economy and posed new challenges to people's livelihood."
Against this backdrop, the Centre believes it is of utmost importance for the Government to put in place corresponding policies to tackle the most pressing issues arising from an ageing population, rising costs of living and the soaring property market.
According to the population figures released by the Census and Statistics Department, the proportion of elderly population, i.e., those aged 60 and above, is projected to rise from 19% in 2011 to 36% in the next 30 years. To mitigate the adverse impact of an ageing workforce, the Centre advocates raising the tax allowances for those aged 60 or above who are assessed to salaries tax or have elected personal assessment from HK$120,000 in the fiscal year of 2012/2013 to HK$150,000 or even HK$180,000.
Under the Centre's proposal, a taxpayer aged 60 or above may claim allowable deductions for their contributions to personal health insurance of up to HK$12,000, as well as expenses for residential care, community care services, dental care and self education with a cap set at HK$76,000, HK$60,000, HK$8,000 and HK$5,000 respectively.
These measures not only help meet the diverse needs of senior people, but also offer incentive for them to continue contributing to the workforce, and in the long run, easing the negative impact of a shrinking labour force on Hong Kong competitiveness.
On assistance for first-time home purchasers, the Centre has all along advocated a shared ownership scheme to aid households whose monthly income is just above the income limit of Public Rental Housing Waiting List and below that of the former Sandwich Class Housing Scheme.
According to the Centre's suggestion, eligible applicants may purchase 50% of a flat and co-own the premises with an intermediary designated by the Government. The applicant will pay a concessionary rent for the other 50% of the flat, and has the option to buy back this 50% at the original price within a fixed period of time. The rent already paid, or part of it, will form part of the final purchase payment. The scheme may take a variety of forms to meet the needs of the applicants and the ability of the intermediary.
Dr Li said, "Most young respondents to our housing survey in July considered 'soaring property prices' and 'insufficient personal savings for downpayment' the main hurdles to their home purchase plans. Our recommendation could offer an immediate helping hand to first-time home buyers. Nevertheless, the Government still needs to match the supply of land for affordable flats to the demand in the market in the long run."
With regard to rising costs of living, the Centre reiterated the importance of a travel cost subsidy scheme to alleviate inflationary pressure. We recommend setting up a multi-mode monthly expenditure cap, applicable across various public transport modes, on qualifying trips made by regular long distance travellers. As the intention is to benefit long distance travellers, it is proposed that only trips (which may include feeder trips on different modes made within a proposed time period, i.e., 90 minutes) costing $14 or above should be counted under the scheme.
The money cap will be triggered once the monthly transport expenditure for such trips exceeds HK$700. Further transport expenditure will be paid by the Government through the Octopus Card until it reaches HK$1,000, while the passengers will have to pay for any subsequent travel cost. The scheme is proposed to be non-means tested as its targets are not just low income groups and is expected to cost the Government about HK$1.1 billion to HK$1.4 billion a year.
"The Government should provide subsidies to make long distance travel more affordable so that people will become more willing to move away from the already densely populated urban areas to outer zones for the sake of sustainable development," added Dr Li.


text: Bosistos Eucalyptus Oil has a distinctive clean fresh fragrance which is stronger and longer lasting, than other eucalyptus oils. Bosisto's Eucalyptus Oil has a wide range of personal and household uses. Eucalyptus oil helps relieve the symptoms of colds and flu, helps relieve muscular and arthritic pain, removes stubborn stains and grease marks, natural, cleaner and freshener, excellent in laundry wash.

subset: commoncrawl


text:
11:14 pm
Shares of 3M Co fell 11 percent on Thursday after the U.S. manufacturing conglomerate cut its 2019 earnings forecast and said it would lay off 2,000 workers, citing worsening performance in high-growth markets such as China.
The stock`s worst day in more than three decades dragged down the Dow Jones Industrial Average, and added to the gloom across the manufacturing sector following bleak results from Caterpillar Inc and United Parcel Service Inc.
3M, which makes everything from adhesive tapes to air filters, said the dismal performance in the first three months of 2019 was caused by weak demand in the China automotive and electronics sectors, causing it to cut production.
"We didn`t respond aggressively enough to what we were seeing. And so, we`re behind the curve," Chief Executive Officer Mike Roman said on a post earnings call with analysts.
Roman added that the maker of Post-it notes and Scotch tape was slow to align its spending with reduced output, and this dented margins.
An analyst on the call peppered management with questions on Roman`s management style and asked if 3M needed to change its incentives or compensation schemes to get its leaders to adapt faster to the changing macro economy.
"We`re going to take stock of what we`re doing, how we`re doing it, and compensation is an important piece and we look at that as well," Roman said.
3M`s shares have underperformed peers such as Honeywell International Inc since Roman took the helm in July last year, with the stock rising 11.4 percent, compared with a 24 percent gain for Honeywell.
"Since the prior CEO stepped down, 3M has really not performed anywhere close to its industrial peers," Melius Research analyst Scott Davis told CNBC.
The results come a day after heavy equipment maker Caterpillar Inc flagged tepid sales in its construction equipment business in the Asia-Pacific region, pointing to subdued growth in China.
After a sharp slowdown in the fourth quarter, China`s economy unexpectedly grew at a steady 6.4 percent pace in the first quarter, but analysts cautioned that it was too early to call a sustainable turnaround.
Sales in Asia-Pacific, 3M`s biggest market outside the United States, fell 7.4 percent, while Europe, Middle East and Africa reported declines of 9.4 percent. Sales in the United States rose just 0.1 percent.
"The only positive that may come out today for the stock is that one of the sell-side bulls may downgrade, as even the most disingenuous analyst has to admit a change in thesis," J.P.Morgan analyst Stephen Tusa wrote in a note.
3M now expects 2019 adjusted earnings between $9.25 and $9.75 per share, versus its prior forecast of $10.45 to $10.90.
The job cuts would result in an estimated annual pre-tax savings range of $225 million to $250 million, with $100 million in the remainder of 2019, the company said in a statement https://www.businesswire.com/news/home/20190425005428/en/3M-Reports-Firs....
(This article has not been edited by Zeebiz editorial team and is auto-generated from an agency feed.)
Get Latest Business News, Stock Market Updates and Videos; Check your tax outgo through Income Tax Calculator and save money through our Personal Finance coverage. Check Business Breaking News Live on Zee Business Twitter and Facebook. Subscribe on YouTube.


text:
CODEC Presents Wretch 32
CODEC Presents Wretch 32
CODEC Presents Wretch 32 Yes thats right… Wretch 32, one of the UK's most celebrated MCs is joining us right here at #CODEC
He has one of the most famous "Fire in The Booths" in history, Check it out here - https://www.youtube.com/watch?v=TPuobIil7-0
As well as some huge tracks that have hit the UK by storm:
'Tell Me' ft. Kojo Funds, Jahlani - https://www.youtube.com/watch?v=XO273RIGifY
'Don't Go' - https://www.youtube.com/watch?v=bj1BMpUnzT8
'Traktor' - https://www.youtube.com/watch?v=MCivYv4HqiI
'6 Words' - https://www.youtube.com/watch?v=DGIYXuZIWBY
Expect nothing less than 2000 people - Three rooms of music - 1 huge party!
//Advance tickets from just £6
//Stage 1 : Wretch 32
//Stage 2 : R&B & Hip Hop
//Stage 3 : Party Anthems
//Drinks start at just £2
Fancy Celebrating in style? For VIP bookings contact email@example.com
Celebrating a Birthday this week? Contact firstname.lastname@example.com to get yourself the best birthday deals in town!
SWX Bristol
Event Type
Club NightView More


text:
You know those people that get to the gym by 6am and are done with their workouts before you have even brushed your teeth? Yeah… how do you be more like them?
A lot of people ask me how they can become a morning exerciser. The simple answer is, wake up early and go workout. But, I know it's not as easy as it sounds.
First, decide if you even want to workout in the morning. Not everyone has the ability to have an effective morning workout. If you are fitting your workouts in after work and having good training sessions, don't mess with a good thing. If you are struggling to get your workouts in, feel tired after a long day, and aren't having good lifts, maybe it's time for a change.
Sleep is going to be the key factor in early workouts. Adults need between 7-9 hours of good sleep every night. Some people can survive off less and some people need more. But on average this is how much sleep you should shoot for each night.
So if you want to wake up at 5:30am to workout, you need to plan accordingly and try to be in bed no later than 10pm.
If that bedtime seems unimaginable to you, ease into it. Adjust your schedule and try to get to bed 10-15 minutes earlier each night until you've reached a bedtime you think you can handle.
Once you have the sleep schedule figured out, it is important to maintain that sleep schedule. You need to be as consistent as possible with your sleep. Even on your off days you should try to be in bed at the same time. You don't want to experience "social jetlag." Similar to normal jetlag, social jetlag will throw off your sleep schedule for days. One night out can leave you with a week of social jetlag. This will throw off your body and make it hard to find that normal sleep schedule you created for your morning workouts.


text:
The SimulScan Document Capture is a software solution that enables customers to extract and process various types of information from documents. A single document may contain barcodes, text, image data and forms, using SimulScan Document Capture this data can be extracted programmatically using OCR, barcode decoding and image capture. For example, a driver's license can be thought of as a template containing regions such as name and address, signature and photo, each of these regions are processed and returned to RhoElements separately by SimulScan Document Capture so Optical Character Recognition captures the owners name and address whilst their photo is captured separately. These regions are defined by a template which maps the various regions of the document to the data they contain and can be created on-line at The SimulScan Document Capture template builder. This API is available only on Symbol Android devices with firmware supporting SimulScan Document Capture.
In order to use this API you must include the following extension in your
extensions: ["simulscan"]
Be sure to review the JavaScript API Usage guide for important information about using this API in JavaScript.
Be sure to review the Ruby API Usage guide for important information about using this API in Ruby.
Capture a document by either taking a photo of it and process that image for fields defined in the template. You must specify the template associated with the capture either through the template property or as part of the propertyMap when calling this method. If the document processing does not succeed the returned callback will contain failure description. If the captured document is processed successfully a callback will contain document data.
Async Callback Returning Parameters: HASH
Indicates whether the document was decoded successfully or not.
Possible Values :
The document was decoded and processed successfully and this callback contains information about the processed form. Look at processedForm parameter.
An error has occurred whilst processing the document. Look at failureReason parameter.
The processing is over. No more notifications until next captureDocument.
Describes reason for failure.
Possible Values :
SimulScan Document Capture engine generic error.
Identification timeout. Refer to identificationTimeout property.
Processing timeout. Refer to processingTimeout property.
The name of the template was used for recognition.
The number of the template was used for recognition.
The name of the template was used for recognition.
The image of whole scanned form.
Width of scanned form in pixels.
Height of scanned form in pixels.
Id of scanned form image. Use getDataUri method to retrieve image data. The id is valid until close method is called.
The name of the group this region is found in, as defined in the template associated with the recognized region. It's optional parameter.
The name of the region, as defined in the template associated with the document.
The number of the region, as defined in the template section associated with the recognized region.
The type of region which has been decoded.
Possible Values :
Optical Character Recognition. The region is a text field. The processedData parameter contains array of lines.
Optical Mark Recognition. The region is a check box or radio box. The processedData parameter of this callback will contain a boolean value.
The defined region was captured as a picture. No processedData parameter. Use the
image callback property with the getDataUri() to get a DataURI of the image.
The region is a barcode and the decoded barcode data is given in the processedData parameter.
The processed data contained in the specified region, this will depend on the type of region being captured. See the values of the
processingMode parameter.
The image of region.
Width of region in pixels.
Height of region in pixels.
Id of region image. Use getDataUri method to retrieve image data. Id is valid until the close method is called.
Relative OCR confidence for processed data if applicable.
Absolute OCR confidence for processed data if applicable.
Synchronous Return:
Method Access:
myObject.captureDocument(CallBackHandler callback)
Rho.SimulScan.captureDocument(CallBackHandler callback)
Rho::SimulScan.captureDocument(CallBackHandler callback)
Close SimulScan Document Capture engine, and release allocated resources. When capturing images, this is especially important since they are made available temporarily.
Synchronous Return:
Method Access:
Fetch templates from dedicated SimulScan Document Capture server and place into template directory. See templateDirectory property. The device must have access to the network and can access the SimulScan Document Capture Template Builder Website
Username of the user.
Password of the user.
Synchronous Return:
Method Access:
myObject.fetchTemplates(STRING userName, STRING password)
Rho.SimulScan.fetchTemplates(STRING userName, STRING password)
Rho::SimulScan.fetchTemplates(STRING userName, STRING password)
This method will return all of object/value pairs for the propertyNames of the API class.
Async Callback Returning Parameters: HASH
Synchronous Return:
Map of all available properties: this method also supports async callbacks - check the Callback tab for callback return parameters.
Method Access:
myObject.getAllProperties(CallBackHandler callback)
Rho.SimulScan.getAllProperties(CallBackHandler callback)
Rho::SimulScan.getAllProperties(CallBackHandler callback)
Return data URI encoding of the raw image associated with the region or captured form, this allows the operator to easily verify that the captured data is accurate and if necessary perform any post processing changes by hand. To show the image on the HTML page insert the data URI string into the src attribute of the img tag:
<img src="dataURI string;">
Id of image to retrieve. Ids are valid until close method call.
Synchronous Return:
Method Access:
myObject.getDataUri(INTEGER imageId)
Rho.SimulScan.getDataUri(INTEGER imageId)
Rho::SimulScan.getDataUri(INTEGER imageId)
This method will return an object that represents the default instance of the API Class. For example Camera.getDefault will return a Camera object that represents the default camera.
Synchronous Return:
Default object of Module.
Method Access:
This method will return a set of object/value pairs for the list of the propertyName that is passed in. The propertyNames must be a valid property of the API class.
List of properties I want to know about
Async Callback Returning Parameters: HASH
Synchronous Return:
Map of properties I want to know about: this method also supports async callbacks - check the Callback tab for callback return parameters.
Method Access:
myObject.getProperties(ARRAY arrayofNames, CallBackHandler callback)
Rho.SimulScan.getProperties(ARRAY arrayofNames, CallBackHandler callback)
Rho::SimulScan.getProperties(ARRAY arrayofNames, CallBackHandler callback)
This method will return the value of the propertyName that is passed in. The propertyName must be a valid property of the API class.
The property to return info about.
Async Callback Returning Parameters: STRING
Synchronous Return:
The property to return info about.: this method also supports async callbacks - check the Callback tab for callback return parameters.
Method Access:
myObject.getProperty(STRING propertyName, CallBackHandler callback)
Rho.SimulScan.getProperty(STRING propertyName, CallBackHandler callback)
Rho::SimulScan.getProperty(STRING propertyName, CallBackHandler callback)
This method allows you to set the attributes of the default object instance by passing in an object of the same class.
An instance object that is of the same class.
Synchronous Return:
Method Access:
Rho.SimulScan.setDefault(SELF_INSTANCE: Rho::SimulScan defaultInstance)
Rho::SimulScan.setDefault(SELF_INSTANCE: Rho::SimulScan defaultInstance)
This method will set the values of a list of properties for the API class. The propertyName must be a valid property for the class and must also not be read only.
Map of properties I want to set
Synchronous Return:
Method Access:
myObject.setProperties(HASH propertyMap)
Rho.SimulScan.setProperties(HASH propertyMap)
Rho::SimulScan.setProperties(HASH propertyMap)
This method will set the value of a property for the API class. The propertyName must be a valid property for the class and must also not be read only.
The one property name that I want to set
The one property value that I want to set
Synchronous Return:
Method Access:
myObject.setProperty(STRING propertyName, STRING propertyValue)
Rho.SimulScan.setProperty(STRING propertyName, STRING propertyValue)
Rho::SimulScan.setProperty(STRING propertyName, STRING propertyValue)
Whether or not to provide audio feedback to the user following document processing.
Default: true
Property Access:
If true, form will be captured automatically when detected. If false, user must manually tap screen or press trigger to capture the form.
Default: true
Property Access:
If enabled, allows a session to write form capture, region images, region values, and other data to storage.
Default: false
Property Access:
Specifies the decode sound (beep) that is heard when a form is decoded.
Default: system/media/audio/notifications/decode.wav
Property Access:
If true, the success event will be sent only after the SimulScan Document Capture dialog is dismissed. If false, the event will be sent immediately after the results are ready. A false value is recommended unless direct interaction with the underlying application is necessary (e.g. keystroke input).
Default: false
Property Access:
Flash mode (off, on, disabled).
Default: off
Possible Values (STRING):
Turn flash off.
Turn flash on.
Disable flash.
Property Access:
Only used if autoImageCapture is enabled. The number of correct frames that is needed to pass before SimulScan Document Capture will automatically start to process form. This should be between 5 and 30.
Default: 15
Property Access:
Whether or not to provide haptic feedback to the user following document processing.
Default: true
Property Access:
The length of time, in milliseconds that the processing engine has to recognize the image from the provided template. From 0 to 20000 in increments of 100.
Default: 15000
Property Access:
Defines where the document should be captured from. The only supported source is camera.
Default: camera
Possible Values (STRING):
The document will be captured from the device camera. After calling the captureDocument method the camera preview will be presented, the user should place the document to be captured in the preview frame and press the soft button to capture & process the image.
Property Access:
Whether or not to provide LED feedback to the user following document processing.
Default: true
Property Access:
Specify which directory to search for logs in.
Default: /sdcard/SimulScanLog
Property Access:
Only used if autoImageCapture is enabled. Number of incorrect frames that is allowed to pass before the frame counter is reset for automatic image capture. This should be between 0 and 20.
Default: 2
Property Access:
Amount of time in milliseconds to wait before timing out processing.
Default: 10000
Property Access:
The template XML which defines the document to be processed. This should be file URI to the template on the device. You must provide this property to define the form being captured and what is contained in each region on that form. Templates are specific to each document format you intend on capturing and can be created on-line at https://dpx-uat.motorolasolutions.com/.
Property Access:
Specify where to store downloaded templates. See fetchTemplates()
Default: /sdcard/templates
Property Access:
If enabled, shows a UI confirmation with results in SimulScan Document Capture View before sending results back to application.
Default: true
Property Access:
Version of SimulScan Document Capture engine.
Property Access:
Before you are able to process any documents with the SimulScan Document Capture engine, you must have template files loaded on the device. These template files must be located in an area that is open to both the SimulScan Document Capture Engine as well as your application. To retrieve the templates from the server, you would use the
fetchTemplates method. This method will communicate with the SimulScan Document Capture back-end and download the templates to the folder that is specified by the templateDirectory property
function downloadSimulScanTemplates(user,password){ // If you include your SimulScan Document Capture template files in the applications public folder // the SimulScan Document Capture engine will not be able to access them due to Android // security policies Rho.SimulScan.templateDirectory = 'file:///sdcard/Templates'; Rho.SimulScan.fetchTemplates(user,password); templates = Rho.RhoFile.listDir(Rho.SimulScan.templateDirectory); for (var i = 0; i < templates.length; ++i) { console.log(templates[i]); } }
The only requirement for capturing a document is to have the SimulScan Document Capture template file location set using the
SimulScan.template property. This must be an absolute URI to the locale file. The location must also be in a folder that is openly available to both your application and the SimulScan Document Capture engine (i.e. it cannot be in the applications
public folder)
// POSTAL.XML was exported from the SimulScan Document Capture Template Builder // In the Template Builder there were several fields defined Rho.SimulScan.template = 'file:///sdcard/Templates/POSTAL.XML'; // Once the captureDocument method is launched, // the SimulScan Document Capture Engine dialog appears instructing the // user to hold the device steady over the printed label // The callback function will be triggered possibly for // many reasons Rho.SimulScan.captureDocument(function(params){ if(params.callbackType == Rho.SimulScan.SUCCESS ){ // process the returned object: var returnedField = params.processedForm; processSimulScanForm(returnedField); } if(params.callbackType == Rho.SimulScan.FAILURE ){ if(params.failureReason == Rho.SimulScan.FAILURE_IDENTIFICATION_TIMEOUT){ // This means it could not identify the label based on the template used // Possible reasons // -label is the wrong label // -Template is poorly defined // maybe scanned in image for defining was not high enough resolution // -camera cannot focus // This may trigger multiple times and may not warrant display information // in your app - the SimulScan Document Capture engine will prompt the user in most cases } if(params.failureReason == Rho.SimulScan.FAILURE_PROCESSING_TIMEOUT){ // This means it identified the form but could not process it // Possible reasons // -Template is poorly defined // maybe scanned in image for defining was not high enough resolution // -Camera cannot focus or poorly lit // This will trigger once and return focus to your application } } });
Now we can loop through the
processedForm object returned by the
captureDocument method. Depending on how the template was defined will determine the type of information in this object. Typically the template will contain several regions that may be OCR (Text), OMR (Check-boxes), Barcode, Picture. It will only return regions that it was able to be processed.
function processSimulScanForm(SimulScanForm){ var regions = SimulScanForm.regions; for(i=0; i< regions.length; ++i){ switch(regions[i].processingMode){ case Rho.SimulScan.PM_OCR: // The text that OCR translated into an array of lines regionTextArray = regions[i].processedData; //maybe do something with returned confidence // PDXform.absoluteOcrConfidence // PDXform.relativeOcrConfidence break; case Rho.SimulScan.PM_OMR: // Checkbox region either True or False regionBooleanCheckbox = regions[i].processedData; break; case Rho.SimulScan.PM_PICTURE: // nothing in processedData // Image held temporarily until Rho.SimulScan.close() // use the id and the Rho.SimulScan.getDataUri method regionImageURI = Rho.SimulScan.getDataUri(regions[i].image.id); break; case Rho.SimulScan.PM_BARCODE: // Barcode data will be in processedData field regionBarCode = regions[i].processedData; break; } } }


text: Starting May 7th, Local H will begin a 7-night stand at Chicago's Beat Kitchen, culminating on May 13th with the release and performance of their latest studio album, 12 Angry Months (Shout!Factory). Local H will perform a different studio album, in it's entirety, on each night with one night dedicated to B-sides and rarities. Beginning with Ham Fisted and ending with 12 Angry Months, die-hard fans will get the chance of a lifetime to hear the Local H catalog performed live. Each night will have surprise guests and a different opening act.


text:
The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls.
IMPORTANCE We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain β-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, β-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of β-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.
Toxoplasma gondii is a coccidian parasite that causes disseminated infections in persons lacking cell-mediated immunity (e.g., persons with AIDS and fetuses) (1, 2). It usually causes a transient illness in immunocompetent persons, but the parasite persists for life as tissue cysts in muscles and brain. Toxoplasma has three walled forms: oocysts, sporocysts, and tissue cysts (Fig. 1A) (3). Cats, the only host that contains sexual forms of Toxoplasma, shed oocysts in their feces. The oocyst wall, which is made by the zygote after fusion of gametes, has two distinct layers. In the environment, oocysts sporulate to form two sporocysts, each of which have a wall and contain four sporozoites. Humans and other warm-blooded animals are infected with Toxoplasma when they ingest sporulated oocysts that excyst in the intestine, disseminate, and form tissue cysts. Alternatively, humans and other warm-blooded animals become infected when they ingest tissue cysts present in undercooked meat. Regardless of the route of infection, Toxoplasma can cross the placenta and infect the fetus. Additionally, Toxoplasma, which is dormant in tissue cysts, may disseminate when patients lose cell-mediated immunity by poorly controlled HIV or by chemotherapy for organ transplantation.
Figure S1
Copyright © 2012 Bushkin et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Eimeria spp. are one of the most common and important parasites of livestock. Unlike Toxoplasma, they are host specific, and mostly, they parasitize the intestine (4, 5). Losses due to coccidiosis amount to billions of dollars worldwide. Eimeria tenella is the most pathogenic coccidium of chickens, and its entire life cycle is confined to the ceca, which are a pair of outpocketings at the junction of the small intestine and the colon. Medication with anticoccidials and immunoprophylaxis with live attenuated oocysts are used to control coccidiosis in poultry (6, 7).
Unlike Toxoplasma oocysts, Eimeria oocysts contain four sporocysts each with two sporozoites (Fig. 1A). Otherwise, oocysts of Toxoplasma and Eimeria are biologically similar. Oocyst and sporocyst walls of Eimeria and Toxoplasma are autofluorescent in UV light based upon the presence of Tyr-rich proteins that form dityrosines (7, 8). Oocyst walls of Toxoplasma also contain Cys- and His-rich proteins that are homologues of the most abundant Cryptosporidium oocyst wall proteins, which are called COWPs (9, 10).
The present studies originated with the prediction from whole-genome sequences of Toxoplasma and Eimeria of a β-1,3-glucan synthase and two glucan hydrolases (11, 12). β-1,3-glucan is a major component of the fungal cell wall that is recognized by Dectin-1, a lectin on the surfaces of macrophages (13, 14). Dectin-1 is a component of the innate immune system and plays an important role in the natural killer cell response to fungi (15). Inhibitors of glucan synthases (caspofungin, micafungin, and anidulafungin) called echinocandins are used to treat human infections with Candida and Aspergillus (16). The goals here were to determine which walls of Toxoplasma and Eimeria contain β-1,3-glucan (oocyst, sporocyst, and/or tissue cyst), identify structures formed by β-1,3-glucan in the parasite walls, and measure the effect, if any, of echinocandins on Eimeria infections of chickens.
The glucan synthases of Toxoplasma and Eimeria resemble plant, rather than fungal, enzymes.We used reverse transcriptase PCR (RT-PCR) from oocyst RNA to identify the coding sequences of the complete Eimeria glucan synthase gene and most of the Toxoplasma glucan synthase gene. The Eimeria glucan synthase gene is ~12,000 nucleotides long and encodes a 2,687-amino-acid protein coded from 27 exons. The Toxoplasma glucan synthase gene has three extra introns not present in Eimeria and is ~18,000 nucleotides long. The Toxoplasma glucan synthase is nearly the same length as that of Eimeria and shows 53% amino acid identity and 70% similarity with the Eimeria glucan synthase.
The predicted Toxoplasma and Eimeria glucan synthases contain a catalytic domain, which is also present in glucan synthases of fungi and plants (Fig. 1B). This catalytic domain includes numerous transmembrane helices at its C terminus, and there is another set of transmembrane helices that is N terminal to the catalytic domain (17, 18). A domain common to the parasite and plant glucan synthases but absent in fungal glucan synthases is present at the N terminus, suggesting recent common ancestry of the parasite and plant enzymes. In contrast, both parasite glucan synthases contain a stretch of >800 amino acids that is absent in glucan synthases of fungi and plants.
Cryptosporidium parvum and Cryptosporidium hominem, which are coccidian parasites that cause diarrhea in humans and animals, are missing all of the enzymes that make or hydrolyze β-1,3-glucan (19). These observations suggest that the oocyst walls of Cryptosporidium are not homologous to the oocyst walls of Toxoplasma and Eimeria, which contain β-1,3-glucan (see next section).
A Toxoplasma glucan hydrolase has a novel glucan-binding domain near its N terminus and is present in the oocyst wall.Like fungi and plants, Toxoplasma and Eimeria have a glucan hydrolase with a GH81 glycohydrolase domain (e.g., TgGH81 [Toxoplasma gondii glucan hydrolase with a GH81 glycohydrolase domain]) and a glucan hydrolase with a GH17 domain (e.g., TgGH17) (Fig. 1C and D) (18). While TgGH17 and EtGH17 (Eimeria tenella glucan hydrolase with a GH17 domain) genes have a single exon, the TgGH81 and EtGH81 genes have ~30 exons. The glucanase domain of TgGH81 and EtGH81 is relatively divergent from those of plants and fungi and does not contain a C-terminal glucan-binding domain (GBD) that has been identified in the Schizosaccharomyces pombe Eng1 (SpGBD [Schizosaccharomyces pombe GBD]) (20). The glucanase domain of TgGH17 and EtGH17 closely resembles that of plants rather than that of fungi, again consistent with common ancestry of the parasite and plant enzymes. In contrast, TgGH17 and EtGH17 have a unique Cys-rich domain between the N-terminal signal peptide and the catalytic domain (Fig. 1D; see Fig. S1 in the supplemental material). We speculated that this domain might be a glucan-binding domain (TgGBD [T. gondii GBD] or EtGBD [E. tenella GBD]), which we tested by making a maltose-binding protein (MBP) fusion protein in the periplasm of bacteria (where disulfides are made) (21).
The glucan-binding domain of the Schizosaccharomyces pombe glucan hydrolase that contains a GH81 domain (SpGBD) (Fig. 1C) binds to Saccharomyces cerevisiae walls that were deproteinated using NaOH (Fig. 2A). SpGBD also binds to oocyst walls, but not sporocyst walls, of Toxoplasma (Fig. 2B) and Eimeria (see Fig. S2 in the supplemental material). The putative glucan-binding domain of the Toxoplasma glucan hydrolase that contains a GH17 domain (TgGBD) (Fig. 1D and Fig. S1) also binds to NaOH-deproteinated Saccharomyces walls (Fig. 2A) and to oocyst walls, but not sporocyst walls, of Toxoplasma (Fig. 2B) and Eimeria (Fig. S2).
Figure S2
Copyright © 2012 Bushkin et al.
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Polyclonal rabbit antibodies to the glucan-binding domain of the Toxoplasma glucan hydrolase (anti-TgGBD antibodies) bind to the oocyst walls, but not the sporocyst walls, of sporulated Toxoplasma (Fig. 2C). These oocysts were permeabilized by freezing and thawing, as shown by binding of the GalNAc-binding plant lectin Maclura pomifera agglutinin (MPA), which labels glycoproteins in the oocyst and sporocyst walls of Toxoplasma (Fig. 2C). In contrast, the anti-TgGBD antibodies do not bind to the oocyst walls of Eimeria. The latter result is explained by differences in the primary sequence of the glucan-binding domains of the Toxoplasma and Eimeria glucan hydrolases (see Fig. S1 in the supplemental material).
Dectin-1, which is the macrophage lectin that binds to β-1,3-glucan, binds to oocyst walls, but not sporocyst walls, of both sporulated Toxoplasma (Fig. 2D) and Eimeria (see Fig. S2 in the supplemental material). Antibodies to β-1,3-glucan also bind to oocyst walls, but not sporocyst walls, of both Toxoplasma (Fig. 2D) and Eimeria (see Fig. S2).
In summary, these results show that four different reagents (SpGBD, TgGBD, Dectin-1, and anti-β-glucan antibody), as well as antibodies to TgGBD, bind to oocyst, but not sporocysts, walls. These results also confirm that the unique Cys-rich domain near the N terminus of TgGH17 is a novel glucan-binding domain (TgGBD) and suggest that glucan-binding is the likely mechanism whereby the TgGH17 is targeted to the Toxoplasma oocyst wall.
The inner layer of oocyst walls of Eimeria is a trabecular scaffold.Transmission electron microscopic studies used unsporulated oocysts of Eimeria (Fig. 1A) that are much cheaper and safer to produce in chickens than are Toxoplasma oocysts in cats (22, 23). As a starting point, we compared the appearance of the oocyst wall of Eimeria with that of Saccharomyces. When ruthenium red is part of the fixative (24), the untreated wall of Saccharomyces shows relatively subtle differences in the granularity of its inner and outer halves (Fig. 3A), while the untreated oocyst wall of Eimeria has two distinct layers (Fig. 3B). The outer layer of the Eimeria oocyst wall is electron dense, while the inner layer is relatively electron lucent. After sonication, the two layers of the oocyst wall of Eimeria often separate from each other.
Negative staining performed prior to breaking oocyst walls shows that the outer layer is a relatively smooth, continuous structure, upon which oval objects are dispersed (Fig. 3C). In contrast, the inner layer of the oocyst wall is a trabecular scaffold that forms a narrow plane in the absence of sectioning (Fig. 3D). The trabeculae of the inner layer of the oocyst wall are 5 to 20 nm thick and have holes ranging from 5 to 250 nm in diameter. At high magnification, these trabeculae contain fibrils (presumably of β-1,3-glucan), which are for the most part straight and evenly spaced (Fig. 3E). Negative staining of broken Toxoplasma oocyst walls also reveals the presence of trabecular scaffolds, showing that these structures are not specific to Eimeria.
In summary, these results suggest that the continuous outer layer of the oocyst wall forms a permeability barrier, while the inner porous layer of the oocyst wall forms a structural support.
Evidence for β-1,3-glucan in the inner layer of the oocyst wall.Gas chromatography-mass spectrometry (GC-MS) of deproteinated oocyst walls of Eimeria treated with acid to release monosaccharides reveals glucose as the major sugar present (see Fig. S3 in the supplemental material). Zymolyase, which contains a mixture of β-glucanases and protease from Arthrobacter luteus (a yeast-digesting bacterium first isolated from brewery sewage), completely removes the wall of Saccharomyces to form a spheroplast (Fig. 4A) (25). The inner layer of the Eimeria oocyst wall, which contains fibrils of β-1,3-glucan, is almost completely removed by zymolyase, while the outer layer of the oocyst wall becomes ruffled (Fig. 4B). In addition, the trabecular scaffolds seen by negative staining are dramatically reduced by zymolyase treatment.
Figure S3
Copyright © 2012 Bushkin et al.
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Mass spectrometry shows that hexose oligomers released by zymolyase from walls of Saccharomyces and oocyst walls of Eimeria have similar sizes, while they vary slightly in relative abundance by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in Fig. 4C and D. These results then confirm the presence of β-hexose in the oocyst wall.
Fibrils of putative β-1,3-glucan are sometimes prominent after oocyst walls are treated with proteinase K, while the outer electron-dense layer is unaffected (Fig. 4E). Anti-TgGBD antibodies bind to the inner layer of Toxoplasma oocyst walls (Fig. 4F).
In summary, the inner layer of the oocyst wall of Eimeria is extensively removed by zymolyase treatment that entirely removes the wall of Saccharomyces to make spheroplasts. While β-1,3-glucan is present throughout the depth of the Saccharomyces walls, β-1,3-glucan appears to be part of a trabecular scaffold present in the inner layers of the oocyst walls of Eimeria and Toxoplasma.
Inhibitors of fungal glucan synthases (echinocandins) markedly decrease recovery of Eimeria oocysts from infected chickens.Echinocandins are antifungal drugs that target the β-1,3-glucan synthases of Candida and Aspergillus (16). Chickens infected with Eimeria were treated via a wing vein catheter with 10 mg/ml of caspofungin, micafungin, or anidulafungin on day 4 after infection, and animals were euthanized on day 6. While caspofungin has no effect on oocyst recovery from ceca, the number of oocysts recovered is decreased by 71 to 84% by micafungin and by 60 to 86% by anidulafungin (Fig. 5A). We recovered about twice as many oocysts from the ceca of chickens treated with 1 and 3 mg/kg of body weight of micafungin and anidulafungin compared to chickens treated with 10 mg/kg of drug. The oocysts recovered from treated and untreated animals labeled with anti-β-1,3-glucan antibodies and sporulate normally.
The mechanism for how echinocandins decrease oocyst recovery was suggested by histological examination of infected ceca. While Eimeria oocysts with stained walls are frequent in the mucosa and in crypt lumens of untreated chickens (Fig. 5B), walled oocysts are more difficult to find in echinocandin-treated ceca. In ceca of drug-treated chickens, there is also an increase in the number of Eimeria oocysts with large, purple-stained vesicles at their periphery (Fig. 5C and D). These large purple-stained vesicles likely are the same as wall-forming bodies that contain Tyr-rich proteins and other proteins, which are secreted onto the surfaces of zygotes to form the oocyst wall (4, 5).
These results suggest that the glucan synthase of Eimeria, like those of fungi, can be targeted by drugs and demonstrate the importance of β-1,3-glucan in oocyst wall formation. While fungi are killed by echinocandins, these drugs appear to arrest the development of oocyst walls of Eimeria, so that immature cysts remain in the mucosa and fail to be released into the crypt lumen.
Knockout experiments show that glucan synthase is not essential for tissue cyst formation by Toxoplasma in vitro. Tissue cysts that contain bradyzoites (slowly dividing forms) can be labeled with Dolichos biflorus lectin (DBL) (walls) and anti-SAG2Y antibodies (bradyzoites) (Fig. 6B) (26–28). In contrast, in vitro tissue cysts and brain cysts failed to be labeled with anti-β-1,3-glucan antibodies and with antibodies to the glucan-binding domain of the Toxoplasma glucan hydrolase (anti-TgGBD antibodies), each of which intensely labels the oocyst wall of Toxoplasma (Fig. 2). RT-PCR with glucan synthase primers with Toxoplasma tachyzoite and bradyzoite mRNA led to few products, many of which contained unspliced introns.
To rule out the possibility that there may be cryptic glucan synthase activity in bradyzoites in vitro contributing to the tissue cyst wall of Toxoplasma, we knocked out the glucan synthase gene in an RH strain of Toxoplasma in which both the Ku80 and the hypoxanthine-xanthine-guanine phosphoribosyltransferase genes have been knocked out (RH ΔHGPRT ΔKu80) (Fig. 6A) (29). These glucan synthase knockouts (ΔTgGS) form tissue cysts in vitro that are labeled with DBL and anti-SAG2Y antibodies in the same way as nontransformed Toxoplasma (Fig. 6C and D). These results strongly suggest that β-1,3-glucan is not a component of the tissue cyst wall, as has also been shown for sporocysts (Fig. 2). Because Toxoplasma loses its sexual competence quickly when it is passed in culture, we were not able to knock out the glucan synthase gene in a strain of Toxoplasma that infects cats and forms oocysts.
With regard to β-1,3-glucan, Eimeria oocysts appear to be a good model for Toxoplasma, because Eimeria contains similar glucan synthase and glucan hydrolases as Toxoplasma, Eimeria oocyst walls can be labeled with the same set of probes for β-1,3-glucan, and the inner layers of oocyst walls of Eimeria and Toxoplasma are each composed of trabecular scaffolds. We were able to use Eimeria to demonstrate release of glucose oligomers by zymolyase from purified oocyst walls and to show that echinocandins markedly reduce oocyst release from ceca from infected chickens.
The inner layer of the oocyst wall has a trabecular appearance, which suggests that it is a scaffold that supports the outer wall and is not part of the permeability barrier that is formed by the outer layer of the oocyst wall. While a scaffold function for β-1,3-glucan is also important in fungal walls, the trabecular appearance of the inner layer of the oocyst wall is distinct. The importance of β-1,3-glucan to the formation of the oocyst wall is shown by inhibitor studies with micafungin and anidulafungin that blocked oocyst release from chickens infected with Eimeria. While we expected that echinocandin treatment would result in the release of oocysts with defective walls, we found instead that oocysts without a complete wall remained within the intestinal epithelium. Tyr-rich proteins that form dityrosine bonds likely contribute to the stability of oocyst walls, as shown for the spore walls of Saccharomyces (7, 8, 30).
Some negative results here have important implications for our understanding of these parasites and their potential control. The absence of β-1,3-glucan in the walls of sporocysts and tissue cysts suggests the presence of another yet-to-be-identified component in the walls of infectious stages of these Toxoplasma and Eimeria that make them hard, impenetrable, and resistant to stomach acids and intestinal proteases. The absence of β-1,3-glucan in tissue cysts suggests that (i) Dectin-1 is not involved in human innate and acquired immune responses to Toxoplasma and (ii) drugs targeting glucan synthase cannot be used to remove residual cysts from persons previously infected with Toxoplasma. The apparent resistance of Eimeria to one echinocandin (caspofungin) that inhibits fungal glucan synthases suggests the possibility that a glucan synthase inhibitor might be found to prevent or treat Eimeria infections in chickens that would not induce resistance by fungi to echinocandins.
Parasites and animals.All animal work was approved by Institutional Animal Care and Use Committee at Boston University and the USDA. Three-week-old Sexsal chickens (USDA) or 6-week-old chickens (Boston University) were infected with 75,000 sporulated oocysts of Eimeria tenella. Eimeria oocysts were recovered using a blender from ceca of euthanized chickens, filtered through gauze cloth, purified by flotation on saturated NaCl solution three times, washed three times in water, resuspended in phosphate-buffered saline (PBS), and stored at 4°C in PBS for short periods or in 1% K2Cr2O7 in PBS for long periods (22). Some oocysts were sporulated by incubation at 30°C for 48 to 72 h with agitation and then stored at 4°C or used for infections.
To determine whether echinocandins had any effect on oocyst production, we treated two or three chickens each on day 4 after Eimeria infection with 10 mg of caspofungin, micafungin, or anidulafungin per kg of body weight in 500 µl of saline that was injected via a catheter into a wing vein. On day 6 (2 days after drug treatment), the chickens were euthanized and Eimeria oocysts were recovered as described above, counted, and examined by fluorescence and transmission electron microscopy. Two birds each were also treated with 1 and 3 mg/kg micafungin and anidulafungin.
Toxoplasma gondii oocysts were obtained from the feces of parasite-free, 10- to 16-week-old kittens that were infected at the USDA with tissue cysts from the brains of Swiss Webster mice infected with the VEG strain of Toxoplasma gondii (23). The oocysts were separated from cat feces by flotation in sucrose solution. The oocysts were incubated at room temperature in 2% sulfuric acid for 1 week, and sporulated oocysts were stored at 4°C. To obtain unsporulated oocysts, we euthanized infected kittens, and unsporulated oocysts were recovered from rectal contents, floated in cold sucrose solution, and stored at 4°C in 2% sulfuric acid to prevent sporulation. The oocysts were transported cold from the USDA to Boston.
Tissue cysts of Pru strain of Toxoplasma in mouse brains of C57BL/6 mice were a generous gift from Jeroen Saeij of the Massachusetts Institute of Technology.
Molecular biology and biochemistry.A Toxoplasma gene encoding putative glucan synthase (TGVEG_028810) was downloaded from ToxoDB and compared with an Eimeria gene encoding glucan synthase (ETH_00000330) using TBLASTX that highlights amino acid sequences common to each parasite protein (11, 12). Reverse transcriptase PCR (RT-PCR) with numerous primers that spanned groups of predicted exons were used to amplify coding sequences for glucan synthases from mRNA from unsporulated oocysts of Toxoplasma and Eimeria. Predicted glucan synthases were compared with each other and with glucan synthases of fungi and plants, conserved domains and transmembrane helices were identified (17, 18), and sequences common to the parasite and plant enzymes were determined by multiple sequence alignments.
The predicted glucan hydrolase of Toxoplasma that contained a single exon product and a GH17 glucan hydrolase domain (TgGH17) (TGVEG_018140) was compared to the Eimeria glucan hydrolase (Supercontig_1 19950 to 198146). A putative Cys-rich, glucan-binding domain of TgGH17 (TgGBD), which was between the signal peptide and the catalytic domain (see Fig. S1 in the supplemental material), was cloned into the pMAL-p5X (New England Biolabs, Ipswich, MA) vector for expression in the bacterial periplasm (21). A maltose-binding protein (MBP) fusion protein (MBP-TgGBD) was expressed in the periplasm of NEB Express Escherichia coli (New England Biolabs). MBP-TgGBD was conjugated to Alexa Fluor dyes (Invitrogen, Grand Island, NY) and used to label fungal and oocyst walls and to make a polyclonal rabbit antibody (anti-TgGBD) (Strategic Diagnostics, Inc., Newark, DE). The Schizosaccharomyces pombe Eng1 glucan-binding domain (SpGBD) was cloned, expressed, and used for fluorescence microscopy in the same way. A plasmid encoding Dectin-1 received from Gerald Fink of the Massachusetts Institute of Technology was transformed into E. coli. Dectin-1, which formed an inclusion body in the bacterial cytosol, was renatured and labeled with Alexa Fluor dye.
The second glucan hydrolase of Toxoplasma, which contains a portion of a GH81 glycohydrolase domain (TgGH81), was roughly predicted by comparing syntenic sequences of Toxoplasma and Eimeria chromosomes using TBLASTX.
Toxoplasma glucan synthase gene knockout (ΔTgGS) and in vitro tissue cysts.T. gondii of the RH strain, which has a deletion of hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) (30) for use as a selectable marker and a deletion of Ku80 (29) to prevent nonhomologous DNA recombination (RH ΔHGPRT ΔKu80), were used as parental strains for glucan synthase knockouts (ΔTgGS). A knockout construct, which consists of an HXGPRT gene flanked by a 500-nucleotide (nt) sequence upstream of the Toxoplasma glucan synthase gene and 500-nt sequence downstream of it, was cloned into a gateway pTKO2 destination vector (a generous gift of Jeroen Saiej of the Massachusetts Institute of Technology) using LR clonase (Invitrogen) (31). Upstream and downstream sequences were cloned into pDONR vectors using BP clonase (Invitrogen). Five-hundred-nucleotide sequences from the 5′ untranslated region (5′ UTR) and 3′ UTR of TgGS were amplified by PCR. Twenty-five micrograms of the knockout construct was transfected into freshly lysed parasites as previously described (32). Selection began one day after transfection, using 25 µg/ml mycophenolic acid and 50 µg/ml xanthine. After 10 to 14 days, parasites were tested for the absence of the glucan synthase gene by PCR using a sense primer upstream of the deleted area and an antisense primer within the HXGPRT marker and sets of gene-specific primers. Parasites were cloned in 384-well plates, and glucan synthase gene knockout was confirmed as described above. A single cell clone of the ΔTgGS knockout was used in subsequent experiments.
Differentiation of tachyzoites to bradyzoites and tissue cyst production in human foreskin fibroblasts were induced by making the medium alkaline in the absence of CO2 (26). The formation of tissue cysts was confirmed by light microscopy and by fluorescence microscopy using Dolichos biflorus lectin (DBL) and anti-SAG2Y antibodies (27, 28).
Mass spectrometric identification of β-glucan in oocyst walls.Saccharomyces cerevisiae cells and Eimeria unsporulated oocysts were broken using glass beads at 4°C in a bead beater for 2 min and then washed twice in PBS. The oocyst walls were deproteinated by incubation with 1 N NaOH at 90°C for 1 h and washed three times in PBS. Prior to zymolyase treatment, oocyst walls were resuspended in α-amylase buffer (100 mM potassium phosphate [pH 6.5]) and incubated with 400 units of α-amylase at 30°C overnight with shaking to remove starch. The oocyst walls were washed three times in PBS, resuspended in zymolyase buffer (10 mM 2-mercaptoethanol, 50 mM potassium phosphate [pH 7.5]), incubated with 5 units zymolyase (Zymo Research, Irvine, CA) at 30°C overnight with shaking, and washed three times in PBS (25). Alternatively, control organisms were treated with α-amylase only. Partially digested walls were then separated from solubilized material by centrifugation at 13,000 × g for 10 min at 4°C.
The supernatants were removed, dried in a Speed-Vac, and kept at −20°C until analysis by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) or electrospray ionization-mass spectrometry (ESI-MS). Before being subjected to mass spectral analysis, dried Eimeria walls were solubilized in a glass tube with 100 µl of a suspension of dimethyl sulfoxide (DMSO) over crushed NaOH pellets, and incubated at room temperature (RT) for 30 min with vortexing to initiate permethylation. Next, 50 µl of iodomethane was added, and the tube was vortexed. After 1 h at RT, the permethylation procedure was repeated once by adding 100 µl of DMSO-NaOH plus 50 µl of iodomethane and incubating the suspension for another hour. To stop the reaction, 600 µl of water was added, and the permethylated carbohydrates were extracted with 1 ml of CHCl3. The organic phase was recovered, washed five times with 2 ml CHCl3-H2O (1:1), and dried under vacuum. The permethylated samples were dissolved as described above and spotted on a MALDI-MS target plate after being mixed 1:1 with a 10-mg/ml 2,5-dihydroxybenzoic acid (DHB) matrix that had been dissolved in the same solvent. MALDI-TOF mass spectra were obtained on a Bruker Daltonics Reflex IV (Bruker Daltonics, Billerica, MA) mass spectrometer operated in the positive-ion reflectron mode. The spectra resulting from 150 and 200 shots from a 337-nm nitrogen laser were summed. The laser pulse width was 3 ns.
For monosaccharide analysis, walls of E. tenella that had been deproteinated by treatment with NaOH were dissolved in 500 µl of 1 M methanolic-HCl at 80°C for 16 h. Sugars were trimethylsilylated in 200 µl of N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 80°C for 45 min. GC-MS analysis was performed on a 6890 series GC-MS system instrument (Agilent, Santa Clara, CA). Sample retention times and mass spectral fragmentation patterns were then compared with those of hexose standards.
Fluorescence microscopy.Toxoplasma and Eimeria oocysts were purified on a three-step CsCl gradient (ρ = 1.05, 1.1, and 1.15) by ultracentrifugation at 16,000 × g for 1 h, washed in PBS, and permeabilized by four cycles of freeze-thawing. Permeabilized oocysts were incubated with 2 µg/ml Alexa Fluor-labeled Dectin-1, SpGBD, or TgGBD in PBS plus 1% bovine serum albumin (BSA) for 1 h at RT and then washed three times in PBS. Alternatively, oocysts were incubated with rabbit anti-β-glucan (a gift from Gerald Fink) or anti-TgGBD antibodies, washed twice in PBS, and then incubated with Alexa Fluor-labeled anti-rabbit antibodies for another hour and washed three times. Alexa Fluor-labeled Maclura pomifera agglutinin (MPA), which binds to sporocyst walls, and cyanovirin-N, which binds to sporozoites, were used at 2 µg/ml to verify permeability (9, 33). In cases where Eimeria oocysts were deproteinated, oocysts were incubated for 1 h in 1 N NaOH at 90°C, washed three times in PBS, and labeled as described above.
Toxoplasma gondii wild-type and glucan synthase knockout (ΔTgGS) tissue cysts grown on glass coverslips were fixed in 4% paraformaldehyde for 30 min at RT, washed three times in PBS, permeabilized with 0.1% Triton X-100 for 10 min, and then washed three times in PBS. Tissue cysts were incubated with 2 µg/ml of anti-SAG2Y antibody (a gift from John Boothroyd of Stanford University) in 1% BSA in PBS for 1 h at RT, washed three times in PBS, incubated with Alexa Fluor-labeled anti-mouse antibody in 1% BSA in PBS for another hour at RT, and then washed three times in PBS (28). Alternatively, tissue cysts were fixed and permeabilized in methanol for 10 min, washed three times in PBS, incubated in 1% BSA in PBS for 1 h at RT with Alexa Fluor-labeled DBL at 2 µg/ml, and then washed three times in PBS (27). The nuclei of host and Toxoplasma cells were labeled by 2 µg/ml DAPI (4′,6′-diamidino-2-phenylindole) in PBS for the last 10 min of antibody or lectin labeling.
Slides were examined by three-dimensional multiple-wavelength fluorescence microscopy using an Olympus IX70 microscope equipped for Deltavision deconvolution (Applied Precision, Issaquah, WA). This system employs restorative and deconvolution techniques. The images were collected at 0.2-mm optical sections for the indicated wavelengths and were subsequently deconvolved using SoftWoRx (Applied Precision). Data were examined either as optical sections or as a projection of the entire stack.
TEM.For negatively stained transmission electron microscopy (TEM), Eimeria and Toxoplasma unsporulated oocysts were either left untreated or treated with zymolyase. For untreated samples, unsporulated oocysts were broken with glass beads in a glass bead beater for 2 min at 4°C with protease inhibitor cocktail (Roche Applied Science, Madison, WI) and then washed three times in PBS. For zymolyase treatment, unsporulated oocysts were broken and washed as described above, resuspended in α-amylase buffer incubated with 400 units of α-amylase at 30°C overnight, washed in PBS, incubated with 5 units of zymolyase at 30°C overnight, and washed in PBS.
The samples were then incubated in 0.1% ruthenium red for 5 min, loaded onto carbon-coated EM grids, washed in 20 mM Tris, stained with 1% uranyl acetate (UA) for 5 min, washed in Tris, and then stained again with UA. Alternatively, in order to visualize the outer layer alone, unsporulated oocysts were first incubated with ruthenium red, washed three times in PBS, stained with UA, washed three times in PBS, and then broken and washed as described above before they were loaded on grids. Images were recorded at 120 kV in a Philips CM12 transmission electron microscope on Kodak SO163 film and scanned on a Nikon 9000 scanner.
For thin-section TEM, Eimeria unsporulated oocysts and Saccharomyces cells were left untreated, sonicated (Eimeria), or broken with glass beads at 4°C (yeast), before washing with PBS and deproteinating with 1 N NaOH at 90°C for 1 h. Alternatively, intact yeast and sonicated oocysts were treated with 5 units of zymolyase at 30°C overnight and washed in PBS. Alternatively, sonicated oocysts were incubated with 40 µg proteinase K (Invitrogen) in 250 µl PBS at 50°C overnight and then washed in PBS.
Treated and untreated oocysts and yeast were fixed in 2.5% paraformaldehyde, 5% glutaraldehyde, and 0.06% picric acid in 200 mM cacodylate buffer for 1 h. Organisms were postfixed for >12 h at 4°C in 1% osmium tetroxide in the presence of 0.1% ruthenium red in cacodylate buffer (24). Water was removed from pellets in a graded series of solutions of ethanol and propylene oxide, and organisms were embedded in Epon at the Harvard Medical School Electron Microscopy Facility. Ultrathin sections (60 to 80 nm) were cut on a Reichert Ultracut-S microtome, picked up and placed on copper grids, and stained with 1% UA and 0.2% lead citrate, and images were recorded at 80 kV in a JEOL 1200EX transmission electron microscope using DITABIS digital imaging.
For immunoelectron microscopy, oocysts of Toxoplasma were fixed in 2% paraformaldehyde and 0.1% glutaraldehyde, dehydrated, and embedded in LR White resin. Ultrathin sections were picked up on copper grids, which were incubated with rabbit anti-TgGBD antibodies. Rabbit antibodies were localized using protein A-gold. Control incubations omitted the anti-TgGBD antibody.
We thank Rudi Beiler and Kath Hardcastle of the Boston University Lab Animals Center for help with wing vein injections. We thank Raymond Fetterer and Ruth Barfield of the USDA for their guidance in Eimeria chicken infections. We thank Maria Ericson of the Harvard Medical School for help with electron microscopy.
This work was supported in part by grants from the National Institutes of Health (NIH) (grant AI48082 to J.S., grant AI07642 [T32] to G.G.B., grants RR010888, GM104603, and RR015942 to C.E.C., grant AI081924 to M.-J.G., and grant GM31318 to P.W.R.). This work was also supported in part by the United States Department of Agriculture (USDA) (CRIS 1265-31320-075-00D to K.B.M.).
- Received 26 July 2012
- Accepted 22 August 2012
- Published 25 September 2012
- Copyright © 2012 Bushkin et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.


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8 Best Places to Work as a Developer In Seattle
Seattle is quickly earning a place as a tech hotspot for young developers, startups, and entrepreneurs. Reports show that Seattle is one of the top markets for tech in the nation, with tech roles accounting for 93 percent of all office jobs added in 2015 and 2016.
Clearly, the city is hungry for tech talent—making it the perfect place for developers looking for new careers. Here's a quick rundown of a few companies you might want to look into.
1. Docusign
Glassdoor rating: 4.6 – 833 Reviews
DocuSign is the premier company in the contract management game, and for good reason. Hailed as a billion-dollar unicorn, DocuSign went public earlier this year with an impressive IPO and recently acquired SpringCM in an effort to compete better against heavy hitters like Oracle and Adobe. Its reviews are stellar, and most of the complaints employees have relate to the company's growing pains. We highly recommend checking them out.
2. Convoy
Glassdoor rating: 4.4 – 44 Reviews
Convoy is all about tech-powered trucking—mobile apps, onboard telematics, transportation logistics. While working in the trucking industry doesn't sound like the sexiest choice for a young developer, don't underestimate them! Convoy was awarded Startup of the Year in the 2018 Geekwire Awards and has been backed by the CEOs of Amazon and Salesforce.
3. PayScale
Glassdoor rating: 4.2 – 124 Reviews
PayScale is an "employee compensation data firm" that is well-known for giving users the inside scoop on what type of pay they can expect at a given job. Current employees praise its unique worker compensation incentives, and with Seattle Business magazine giving it a top spot in its 100 Best Companies to Work For in 2018, it's clear they're doing something right.
4. RealSelf
Glassdoor rating: 4.3 – 106 Reviews
RealSelf is a known entity in community-based health information, wellness tips, and cosmetic surgery knowledge. The company generates revenue by giving medical clinics targeted exposure for services, particularly in the fields of plastic surgery and other non-surgical procedures. If this sounds like an option that's up your alley, now might be the perfect time to jump on board—the company recently raised $40 million in seed funding to boost its brand on an international level.
5. Redfin
Glassdoor rating: 3.3 – 455 Reviews
Seattle's growing role in the tech industry has insulated the city somewhat against the declining housing market. And while real estate companies like Redfin are more susceptible than others to fluctuations in the economy, the company is making big moves in cutting out the middleman in real estate and making the process of closing on a home entirely digital. For that reason alone, we expect it to weather the storm.
6. Rover
Glassdoor rating: 4.5 – 86 Reviews
You wouldn't think a pet-sitting startup would be able to generate $65 million in funding after only six years of operation, but then you saw what happened to Rover in 2017. The company has seen substantial growth over the past few years, with the company claiming that a service is booked through Rover every four seconds. Rover is open about its need for talent and is actively recruiting, so make sure you check it out.
7. Textio
Glassdoor rating: 5.0 – 12 Reviews
Here's something different: Meet Textio, a new augmented writing platform designed to help companies write better job postings. Though the company is only a few years old, its forward-thinking nature and commitment to inclusive hiring have helped them raise nearly $30 million in funding, earning them a top spot in Seattle Business magazine's 100 Best Companies to Work For in 2018.
8. Highspot
Glassdoor rating: 4.9 – 51 Reviews
Highspot provides AI-based sales solutions to help companies better reach their customers, with clients that include Amazon, DropBox, and Twitter. Highspot is in a lucrative market at a pivotal point in the industry's development—AI is getting big and becoming a desirable choice for developers of all kinds. The company has earned $64 million in funding since its inception, so make sure you check them out.
Not seeing anything that piques your interest? Don't worry. Seattle has tons of tech jobs available if you know how to find them. If you're struggling to find a job that suits your talents, feel free to contact Urgenci and we'll give you a hand.
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Montessori Wooden Counting Toy by Olly&Owl
$69.99 $44.99
Use these geometric toys to help them learn! With shapes, colors, and numbers! ⭕ Easy to use and made from wood. Get them prepped for preschool with early-stage development & support.
Hands-on tools always stimulate more engagement. Great toy to always have around between 0 to 36 months as they develop 💠 It is recommended to learn colors first, then shapes and numbers.
- Made from wood & safe for the baby!
- Includes numbers, rings, and objects.
- Increase mental 🧠 skills and motor functions.
- Great for bonding with kids during playtime.
- Lots of pieces to share between multiple kids.
- Environmentally friendly plastic components.
- Teach early math skills.
- Teach colors, shapes, and numbers as they progress.
- Give them a break from their electronic toys and tv!
- Hands-on playtime 😄


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We Give What We Have; Not What Others Need
January 8, 2019
One of the most important truths I have learned in my lifetime is that I can offer others what I have, not all they need. If someone is sad, I can offer to listen, but I can't take away their sadness. If someone has experienced loss, I can empathize, but I can't heal them. Sometimes, the best I have to offer is to be present with their sadness or grief, which feels like so little at the time. Whenever I am in the presence of another's pain, it is hard, yet, I want to offer what I have in hopes of helping.
Years ago I was sitting with a member of a church I was serving; we were in a hospital ER while physicians were attending to her husband, who had just shot himself. I was trying to heal her pain, mainly because I felt so uncomfortable and inadequate in that moment, by spouting off words of wisdom. Later, she informed me that she didn't remember a word I said, but did remember my presence, which offered a little comfort. I offered her what I had; while she needed a lot more, I was able to offer something.
Never Walk Down the Back Alley of Your Mind Alone
January 8, 2019
Hope is often found when we are open to recognizing our needs and asking for help. Hope is often lost when we are isolated and trying to live our lives cut off from others. One mentor of mine once said, "David, never walk down the back alley of your mind alone." We all need help sometimes, especially when we face a crisis.
I have often wondered why it is so hard to ask for help; maybe its because I don't want to look weak or am afraid someone will use my vulnerability against me. In any case, I have learned over the years that when I am at those points where I need help, trustworthy people are more than willing to offer what they have. What I most often needed was simply someone to pay attention to me and what I had to say. There is something soothing about having someone stop what they are doing, make eye contact, and position their body toward me and offer attention. When one of my kids was small and fell and skinned their knee, what they wanted more than Neosporin and a band aid was my warm attention and touch with, "Oh my, that must have hurt; I am right here and not going anywhere."
When I receive appropriate attention, I calm down and hope rises within.


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Create an Account - Increase your productivity, customize your experience, and engage in information you care about.
Discover what classes and events are offered by the library.
Find information on how to obtain an inter-library loan.
Utilize the materials request form to obtain a needed item.
Review important tax and government related information.
View potential volunteer opportunities available.
We love our community and its food! Let's bake some things that remind us of home together! We'll meet up and eat up on Saturdays at 10am via Zoom.


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Conceptual Review of Pharmacology for NBE
Conceptual Review of Pharmacology for NBE by Ranjan Kumar Patel is a book useful for all PG medical entrance examination.
Exclusive Features:
- Chapterwise Synopsis with Image based Q's (Integrated Approach)
- More than 2500 Explained Q's with References covering All National level Exams (AI, DNB, AIIMS, PGI,UPSC, JIPMER) & State exams (TNPG, MAHPG, APPG and many more)
- All Q's given Topic wise & Sub topic wise.
- 300 new Q's added from most recent exams of 2014-16
- 100+ conceptual, clinical boxes & Illustrations.
- 100+ Image-based Q's from all recent Exams
- Strong Facebook Support.


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The golden eagle is the largest predatory bird in North America. They prefer to hang out in cliffs along rivers, canyons, mountainous regions, and bluffs; anywhere they can get a good updraft. They are the most widely distributed of all eagles. They range from Mexico all the way up to Alaska. Considering the fact that their eyes don't have a wide range of movement, golden eagles are able to move their heads up to 270°. They have keen eyesight and can see great distances and in color.
First the Stats…
Scientific name: Aquila chrysaetos
Weight: Up to 15 lbs.
Length: Up to 3 feet
Wingspan: Up to 7 feet
Lifespan: Up to 30 years
Now on to the Facts!
1.) These giant raptors are one of only a few that have feathers all the way down to their talons.
2.) Like all raptors, these eagles are carnivores (eat meat).
3.) A golden eagle is powerful and large enough to take down an adult deer! However, they prefer rabbits, hares, small rodents, reptiles, other birds, fish, and carrion (dead animals).
4.) This raptor is able to dive at speeds of up to 150 mph!
5.) These eagles are a protected species but are listed as Least Concern, by the IUCN.
But wait, there's more on the golden eagle!
6.) Mating for life is commonplace with these majestic birds.
7.) A mated pair, being very territorial, can monitor an area of up to 60 square miles!
Did you know…?
The golden eagle's talons can grow up to 3 inches long! Being struck by these talons during a dive has been compared to being hit with a bullet!
8.) Their nests are gigantic and can be as large as 5 – 6 feet across and 2+ feet deep; and are made of sticks, pieces of fencing, wires, and even bones. They sometimes add scented leaves to help keep insect pests away.
9.) The biggest nest on record, for a golden eagle measured 20 feet tall and 8.5 feet wide!
10.) Females lay between 1 – 3 eggs.


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Contact: George Pytlik, The Pocket Testament League, 888-598-3595
LITITZ, Penn., April 11 /Christian Newswire/ -- The Pocket Testament League (www.pocketpower.org) has introduced a new free Gospel of John in the ESV (English Standard Version) translation using a cover design oriented towards the construction industry. Headlined "Blueprint for Life," the wrap-around cover features the photograph of a blueprint, lumber and tools. One of many different cover designs that make it easy for people to share their faith by giving away a free gift, the new layout was especially created for sharing with people in the building trades such as architects, cabinet makers, plumbers, electricians or even clerks in the local hardware store.
League president Mike Brickley says, "Those with a passion for sharing God's Word have a new tool they can use, whether they are remodeling their kitchen, selecting new paint or getting a carpet installed. All they do is give away a free Gospel of John as a gift. It's so easy."
He says that members of the League have given away more than 100 million Gospels in the history of the Christian outreach ministry, forever changing hundreds of thousands of lives. "Almost everyone you share with appreciates the gesture and accepts the Gospel gift with a grateful heart," he says.
Explaining that the League offers free membership and that Gospel booklets are completely free of charge, he adds that "Thousands of members share stories each year describing the impact they are having with these Gospels of John. Again and again we hear how this small gesture of giving away a booklet happened at a critical moment in someone's life, when they most needed to connect with God."
The League's free Gospels of John and other resources are offered in English, Spanish and Chinese. Membership is free. Members may order Gospels every month to give away as special gifts in one-to-one encounters in their day-to-day lives.
More information about The Pocket Testament League Christian outreach ministry is available on their web site at www.pocketpower.org


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Face ID can be used from the age of 13
Developers building app for kids under 13 will have to forget about introducing Face ID as a method of authorization.
After the presentation of iPhone X with 3D face scanner, Apple has published a new list of rules for developers. In it, the company warns that it will not flow into the AppStore apps for kids authorization through a Face ID.
Developers will have to come up with another method of authentication.
The issue of security Face ID bothers many users. Recently, the Senator of the US democratic party, al Franken turned to Apple in an open letter and asked the company to explain how Face ID protects users ' biometric data.
Despite the fact that all data obtained by the scanner is stored on the iPhone, it is quite logical that Apple is trying to protect yourself from possible problems in case of usage of scanner children.
Probably talking about the Federal law on protection of personal data of children under the age of 13. Because of it children under this age are unable to register themselves in social networks, create a Google account to use AppStore.
Technology Face ID was presented together with the iPhone X on September 12, "the Theatre of Steve jobs". In addition to the new smartphone, Apple revealed the Apple Watch and third-generation Apple TV 4K.
Follow the news Apple in the app on iOS MacDigger.


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Leadership Inspirations Activities
Anything Goes
Try to win your argument about anything
10-20 minutes
Team Stage
Storming, Norming
Type Of Activity
Experiential Activity
In this activity, participants must try to win an argument about a truly random topic. Use this activity to explore the difference between debate and dialogue, and agree on effective processes to resolve disagreements or conflicts.
- Break your group up into pairs.
- This activity will begin a little like "Rock, Paper, Scissors", but participants will chant together, "Nothing, Something, Anything"
- After "Anything", each participant should say the name of any random object that comes to mind (turtle, envelope, water bottle etc.).
- Facilitator Note: Remind participants to be appropriate in their choices.
- Participants will try to win a debate about why their object would "win".
- Example: Turtle vs. Water Bottle
- Give them each a few minutes to make their cases.
- Have them pause and lead a mini debrief with the group about their conversations so far with questions like these:
- How is the debate going?
- Who is "winning" and why?
- What are you feeling at this point in the debate?
- What is the difference between a debate and dialogue?
- What would you need in this conversation to see the other person's side?
- Instruct your groups to continue this conversation, but now as a dialogue.
- After a few minutes, have your groups come back together for another debrief:
- What differences did you notice between this conversation and the first?
- What was easier or harder about this conversation?
- In what situations are debates useful to our group? In what situations do we need dialogue?
- How do we want to use this understanding moving forward to work better together?
- For a team version of this activity, try Early Bird vs. Second Mouse
- Use this activity as a warm up for brainstorming and ideation. Come back to these learning points as your group tries to reach consensus.
- What was the goal of this activity?
- Did your group do well? What could have improved?
- How did your group make decisions? Was it effective or ineffective?
- How did your group manage conflict that arose?
So What?
- Are we the only ones that define our purpose? What or who influences our purpose?
- How do we communicate our purpose to new members or people outside of our group?
- Why is it important that we all understand the purpose of our group/activity/etc.?
Group Dynamics
- What are the traits that make someone successful on this team? What are the traits that make someone unsuccessful on this team?
- What motivates our group?
- How does our group dynamic affect the way we get work done?
Meeting Skills
- What are some of our effective and ineffective meeting habits?
- When are you most engaged in meetings? When are you least engaged?
- What are the roles that people play in meetings? Why are these roles important?
Now What?
- What does this activity tell you about the strengths of your group? The areas for improvement?
- What's one commitment each person can make?
- What are three lessons the group has learned that they can continue to work on?
- How can we implement these lessons in our school/organization?
- What can you do differently moving forward?


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A bid to get planning permission for a controversial 41-flat scheme in Middlesbrough has hit a brick wall.
Bett Homes wants to build a four-floor complex on land occupied by a bungalow and the former Nessfield care home at Morton Carr Lane, Nunthorpe.
An appeal was lodged after Redcar and Cleveland Council refused planning permission.
But Government Inspector Peter Jamieson has now dismissed the appeal, saying the proposal would be unsuitable for that area.
However, a new application was submitted by Bett Homes in April.
A 589-name petition by residents against the plan was considered by the inspector.


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Grilling brings out the flavor in a wide variety of vegetables without adding a lot of fat or calories. Use this recipe for a simple and healthy addition to your next cookout.
- Choose a vegetable. Corn on the cob with husks removed, bell peppers cut into large chunks, asparagus, and zucchini sliced lengthwise are great choices.
- Add a little oil and seasoning. Rub the vegetables with just a little extra virgin olive oil or canola oil and sprinkle with salt and pepper. Feeling more adventurous? Add other spices like chili powder or Cajun seasoning, or sprinkle with fresh herbs such as garlic, rosemary, thyme, or terragon!
- Grill 8 – 12 minutes. Heat your grill to medium high and grill the vegetables, turning often, until crisp and tender.


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Post Highlights
- 1 Tripura Police Riflemen Syllabus 2019
- 1.1 Tripura Police Rifleman Syllabus 2019 Details
- 1.2 Check Tripura Police Riflemen Exam Pattern
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Tripura Police Riflemen Syllabus 2019 is now available for the Applicant to download the pdf appliers must refer the ripurapolice.gov.in Syllabus download online links with the sake of the Applicants we also update the Tripura Police Riflemen Exam Pattern to get the examination Practice Session in the perfect way. Actually, this Tripura Police Tradesmen Exam Pattern is very difficult with the use of the pattern there would find the Topic about the examination and also the Marks allocated to each subject was also presented in the pattern. So, just make over and Verify the tripurapolice.gov.in Riflemen Syllabus with the exam structure.
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Tripura Police Rifleman Syllabus 2019 Details
|Name of the Organization
|Tripura Police Recruitment Board
|Name of the Post
|Riflemen & Riflemen (Tradesmen)
|No. of Posts
|Job Category
|Job Location
|Official Website
Tripura Police Riflemen Exam Pattern 2019
|Subject Names
|Exam Duration
|Part A
|General Intelligence & Reasoning
|2 Hours
|Part B
|Elementary Mathematics
|Part C
|English / Hindi/ Bengali
|Part D
|General Knowledge & General Awareness
PST (Physical Standard Test)
|Candidate Category
|Male (UR & SC)
|170 Cms
|80 + 5 Cms Expansion
|Female (UR & SC)
|157 Cms
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Tripura Police Riflemen Syllabus 2019
English Language
- Sentence Completion.
- Active Voice and Passive Voice.
- Sentence Arrangement.
- Joining Sentences.
- Para Completion.
- Fill in the blanks.
- Synonyms.
- Substitution.
- Error Correction (Underlined Part).
- Antonyms.
- Error Correction (Phrase in Bold).
- Spotting Errors.
- Idioms and Phrases.
- Sentence Improvement.
- Prepositions.
- Passage Completion.
General Intelligence and Reasoning
- Analogies.
- Judgment.
- Similarities & Differences.
- Decision making.
- Arithmetical Number Series.
- Space visualization.
- Arithmetical reasoning.
- Visual memory.
- Non-verbal series
- Verbal and Figure Classification.
- Relationship concepts.
- Analysis.
- Problem-solving.
- Average.
- Use of Tables and Graphs.
- Mensuration
- Relationship Between Numbers.
- Discount.
- Time & Distance.
- Profit & Loss.
- Decimals and Fractions.
- Partnership.
- Time & Work.
- Fundamental Arithmetical Operations.
- Percentage.
- Ratio & Proportion.
- Number Systems.
- Computation of Whole Numbers.
- Simple Interest.
General Knowledge & General Awareness
- Indian History.
- Indian Politics.
- General Science.
- Tourism.
- Famous Places in India.
- Environmental Issues.
- Rivers, Lakes, and Seas.
- Heritage.
- Current Affairs.
- Famous Days & Dates.
- Biology.
- Artists.
- Countries and Capitals.
- Inventions and Discoveries.
- Geography.
- Literature.
- Indian Economy.
- Famous Books & Authors.
- Sports.
- Indian Parliament.
- Civics.
Check Tripura Police Riflemen Exam Pattern
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Tripura Police Riflemen Syllabus 2019 Downloading links
Official Website: tripurapolice.gov.in
Download Tripura Riflemen (GD, Tradesmen) Syllabus 2019


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We did crossfit today.
A local place … coast range crossfit has been beyond generous in gifting us a memberships. It's the first time since I have known him that I have seen Tony truly excited about exercising .. I think it will be crucial component of his healing process. Being active was even a "homework" assignment for him from our therapist…
First song for the workout… Brave.
It makes me happy also that he is giving our boys this example of taking care of himself in a multitude of ways. I hope we are able to raise them to be well rounded.. not just despite of our grief.. maybe even because of it.
A friend watched the 3 youngest for us. The same family that came over to play on the water slide. Its sometimes surprising to me what is a trigger and what isn't.
My boys were so happy to be playing with their girls.. a different kind of joy I haven't seen in awhile. The way I felt watching them play, in particular with the daughter that was Jennifer's friend, a classmate from preschool last year… It was like when you go away on a trip and come back ..it just feels good to be home.
I think thats how they felt..
… Content. Comfortable.
..whole. Even if artificially.
.. .happiness in a way I can't provide.
Watching them parade around after her.. just like they did with sissy. Playing the way bossy girls make younger boys play with them. Pink socks on their hands..
Me too. I would have thought seeing that.. hearing that… would have been crippling. But it wasn't. Tony and I just kept saying to each other look how happy they are.. I think for us it was the same.. happy to see them full of sisterly
I would go again tomorrow and the day after that.. it really wasn't hard in the moment. But now.. I hurt.
i cry.
Alone at night.. remembering the happiness not of today but of our yesterdays.
i cry.
Realization that I will never again really be complete.. another layer of what we have lost.
Tonight I somehow can't find the words. I am just looking at the program from her services and aching.
Just so sad.
I used that word a lot before. I think I had no idea the real meaning.. the real power of the word. But here tonight… writing in my pink jammie pants that she got me for Christmas I know the magnitude of those 3 letters.
I just want to hold her. I want to feel her weight on my lap. To feel her arms around me.
I want her to welcome Daddy home from work each night.. waiting outside hiding so he doesn't see or noses pressed against the window.
I want her brothers to follow her around.. I want them to have that simple pleasure again.
I want her baby sister to hold her hands learning how to walk.. .
My kids have lost so much. Today somehow I saw.. I felt .. how much.
I don't just want my daughter back. .. I want my family back. All of us. None of us are the same. Tony, Libby, Jonathan, Nicholas and Charlotte…This disease killed more than just her. It killed the old us. The real us.
I just want to go home.
We are all so changed. Extended family too. My brothers.. too much emotion there for me to write. .. Her cousin without her partner to be playing girlie games with at Easter. My nephew .. unable to run up to make her laugh.. trying now to connect with Jennifer's little sister.
It pains me.. it also isolates me. ..
My boys made me proud tonight. Each having a moment I can look at and see the positive of Jennifer in. Nicholas was being a good bigger guy to our friends younger son. He played with him.. engaged with him.. When Nicholas fell off a chair and got stuck and cried, Jonathan stopped what he was doing to ask if his little brother was ok.
When it was time to leave tonight Jonathan did not want to go. He hunkered into his angry. Kicking toys. Not obeying. He lost both books at his bedtime then.. but thankfully pulled it together just enough so we could still have our snuggle time in bed…
It's hard enough to navigate these years without adding in a murkiness impossible to penetrate with sadness this strong …
We have to discipline. We have to give allowances. And we have to do it all on the fly.
No way to know if we are doing it right.
In his bed we prayed together.. thankful for the help we are receiving as a family from so many avenues. And for the first time that I have witnessed he talked to Jennifer out loud. He was a energy ball tonight going to bed. Just full of emotions bouncing around inside of him..
I get it.. I feel the same a lot of the time. But I get this.. I get to write at night to sort it all out. I know he doesn't even understand how being with the girls was wonderful.. how it made him so happy… but its a double edged sword…it's like it reminded his cells that she is gone.. parts of him activated that haven't been used since a few weeks before she died… probably not since our Make A Wish trip..
.. . he was just starting to settle back into her being around.. Only to have her stolen away forever.
I guess when your child dies you can never really
.. . go home again.
**the fuzziness is perfect in this picture. This is the heavy and the fog of sadness. It's how sad distorts my vision. **


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Death Guard: Plagueburst Crawler
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Product description
A multipart plastic kit that makes 1x heavy artillery vehicle of the Death Guard. Side sponsons can be armed with two different options.
Plastic miniature. Assembly and painting required.


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Event description
Doctors. Scientists. Advocates. Athletes. Changemakers. These are the video and live speakers who will gather at our BRAINx event at Ramada Kelowna on June 7, 2018.
Some of the most inspired thinkers in the field will share short, powerful insights into brain injury. Come for the experience. Stay for the conversation.
This event is made possible through the generous sponsorship of Pushor Mitchell LLP.