text:
Q: error when Trying to read State Names using string tokenizer This program allows a user to calculate to enter sales and calculate sales taxes per state.
The program is giving errors when it gets to reading the double word states (eg New Jersey).I need helo with part.
    import java.text.DecimalFormat;
import java.util.*;
import java.io.*;
import java.lang.reflect.Array;
import java.nio.CharBuffer;
import java.awt.*;
//import java.awt.event.*;
import javax.lang.model.type.ArrayType;
import javax.swing.*;
import javax.swing.event.*;
public class PJ5 extends JFrame implements ListSelectionListener
{String []States = new String[100];
    double []statetax = new double[100];
double sales;
JPanel p1;
JList  StateList;

JTextField salesField;
private JTextField taxField;
public static void main(String []args)
  {
  PJ5  x = new PJ5();
  x.setDefaultCloseOperation(JFrame.EXIT_ON_CLOSE);
  x.setTitle("Sales tax calculator");
  x.setSize(350, 250);
  x.setVisible(true);

  } // main

public PJ5() // constructor
{
String s;
StringTokenizer st;
int k=0;
try
{
BufferedReader inFile = new BufferedReader(new FileReader("C:\\Users\\Owner\\Documents\\taxrates.txt"));
    while ((s= inFile.readLine()) != null)
    {

st = new StringTokenizer(s);
        States[k]=(st.nextToken());

    statetax[k] = Double.valueOf(st.nextToken());

    ++k;
    } // while not EOF
  inFile.close();
} catch (Exception e) { System.err.println(e); }

JPanel p1 =new JPanel();

 p1.setLayout(new GridLayout(2,1));

p1.add(new JLabel("Sales amount:"));
salesField= new JTextField(5);
salesField.setEditable(true);
p1.add(salesField);

p1.add(new JLabel("tax:"));
taxField= new JTextField(4);
taxField.setEditable(false);
p1.add(taxField);

add(p1,BorderLayout.NORTH);
pack();
setVisible(true);

JPanel p = new JPanel();
StateList = new JList(States);  // create a JList object
StateList.setSelectionMode(ListSelectionModel.SINGLE_SELECTION);
JScrollPane sp = new JScrollPane(StateList);  // adding scrolling capability
p.add(sp);
getContentPane().add(p, BorderLayout.SOUTH);
// Event registration
StateList.addListSelectionListener(this);
} // constructor

// Event handling
public void valueChanged(ListSelectionEvent event)
     {
    DecimalFormat dollar = new DecimalFormat("0.00");
    double tax;
     sales=Double.parseDouble(salesField.getText());

tax=sales* (statetax[StateList.getSelectedIndex()]/100);
     taxField.setText("$"+dollar.format(tax));

     } // valueChanged
}

here is the text file with the states en their tax rates.
Alabama 4.0
Alaska 0.
Arizona 5.6
Arkansas 6.0
California 8.25
Colorado 2.9
Connecticut 6.0
Delaware  2.07
Florida 6.0
Georgia 4.0
Hawaii 4.0
Idaho 6.0
Illinois 6.25
Indiana 7.0
Iowa    6.0
Kansas  5.3
Kentucky    6.0
Louisian,   4.0
Maine   5.0
Maryland    6.0
Massachusetts   6.25
Michigan    6.0
Minnesota   6.875
Mississippi 7.0
Missouri    4.225
Montana 0.
Nebraska    5.5
Nevada  6.85
New Hampshire 0.0
New Jersey  7.0
New Mexico  5.375
New York    4.0
North Carolina  4.5
North Dakota    5.0
Ohio    5.5
Oklahoma    4.5
Oregon  0.
Pennsylvania    6.0
Rhode Island    7.0
South Carolina  6.0
South Dakota    4.0
Tennessee   7.0
Texas   6.25
Utah    5.95
Vermont     6.0
Virginia    5.0
West Virginia   6.0
Wisconsin   5.0
Washington  6.5
Washington DC   5.75
Wyoming 4.0

A: Its because, the tokenizer parses New as a string then the next code parses Hampshire as a double, you may need to restructure your code


I read this article with interest and found the position adopted fairly simplisitc, in the way the world is neatly divided into Muslims and non-Muslims. That is certainly an imaginary division. There are close to six million Muslims in America alone. What is deemed as the West cannot equate unequivocally to Christendom, which I believe the writer of the article is aware of. Moreover, the article implies a homogenous Muslim world, which is a sure fallacy for anyone who has lived in different Muslim countries.
Instead of looking at a war between civilizations, I think the problems of Islamic extremism are confined to the few who are fueled with radical nonsense and utilize religion as a tool to gain support. In reality their margin for success is quite narrow. Historically, there were instances of such radicalism early in Islamic civilizations (The Khwarij). Such violence stemming from Muslim radicals must certainly be put to stop.
I believe, Muslims (the term moderate Muslims is ridiculous) should be more active in weeding out such extremisms within their own communities. Furthermore, I would like to reassure the writer of the article that there is no global jihad. If there is, the scale of conflict, I am sure, will be far different.
Admittedly, I am not a fan of Daniel Pipes, but I do agree with his third option of American Troops playing a support role rather than getting in-between sectarian violence. However, the notion of the Clash of Civilizations is certainly, something, that at this point is not going to help make matters better. Yes, there will always be irreconcilable differences between different religions. It does not mean we have to kill one another because of it. Let common sense prevail.


The Gizmo is the extra hand you always need when separating chain-pieced segments of your quilt tops. The rubberized base guarantees that it stands securely and does not slip, allowing you to safely cut threads. Hold the fabric pieces with both hands and slip the thread into the slot on the top of the Gizmo. The embedded razor blade cuts the thread quickly and easily. Why not keep one by your sewing machine and one by your ironing station?


I lay here, curled under the covers at 1 p.m. Motionless, I stare at the curtain blocking the world out. Sometimes I think it ripples, feeling the breeze against the window. The overnight rain has subsided but I guess there's more to come. The dark cloud of depression has settled itself in my room. Stretching out. Getting comfortable. The air feels thinner now. It's a struggle to breathe. In fact, everything is a struggle.
This thick veil of blankets used to weigh me down, but in this moment I think it's my very existence causing undue pressure. I repeat over and over how sorry I am — sorry for the burden I feel I've become, the trouble I seem to cause, the constant worry you shoulder. The fear of not knowing who I'm going to be when you arrive home: angry or agitated or manic or depressed. Or worse yet, cycling through them all.
Some days I feel as though I have lost myself. Deep within the depths of my mind lies truth and judgment. Though I judge no one only myself, as I am the one to cast the very first stone. The utter emptiness and transparent sudden dissociation from all that I once loved has Settled within me so deep that no pill, no alcohol or amount of love could possibly seep through and dissolve it. I have fallen into a pit and I'm searching with everything in me to come out alive. I dream of confinement, I pray for understanding and most of all I hope for peace. In the rollercoaster of emotion I am running from one high to the next. Enjoying new smells, sounds, and tastes. Shopping feverishly to contain the madness but the madness has contained me and all that's left is to just go along with it. Try to minimize as much of the damage as I can. Until I can no longer deal and all that I so looked forward to seems pointless and suffocating.
Natasha Velez born and raised in New York City, is a Financial Controller and Accountant practicing in Central Florida. She suffers with Rapid-Cycling Bipolar Disorder and is a blogger and writer for sites such as The Mighty, The Huffington Post and This Is My Brave. She recently started writing a book on her experiences with mental illness and overcoming the stigmatism that comes with it in the professional industry and her personal life. Her acceptance and passion for fighting the stigma that comes with mental illness is what caused her to become a mental health advocate along with being fiction and non-fiction writer.


Well, well, what do we have here, yet another case of the Pot, the U.S. Congress, calling the Kettle, in this case the VA, black? Suddenly the members of this Congress have awakened from their deep sleep and have launched a massive attack on the VA, long before all the facts and evidence of alleged VA abusive practices have been fully determined.
Watching this latest governmental fiasco enfold in Washington is like watching re-runs of the Keystone Cops. The vast majority of the members of both the House and the Senate, with a few notable exceptions, are classic surface-thinkers and political lightweights that couldn't solve a critical problem if the solution were injected directly into their brains.
How about this for extreme audacity? Here we have one of the worst performing, least respected institutions in America, the U.S. Congress, condemning the VA health care system for gross mismanagement and incompetence. If that's not the pot calling the kettle black then what is it?
But let's get serious and address this difficult, troubling situation based on the facts as we currently know them. Yes, these problems with the VA are real and they have, no doubt, contributed to any number of needless deaths of veterans who did not receive timely treatments. But there is no need to condemn this respected medical institution for alleged negligence and make yet unproven allegations about it that stain its reputation and cover many of its dedicated employees with a blanket of guilt; certainly not until the evidence is in.
There is no doubt that some incompetence exists and questionable methods have been used to cover up deficiencies but, unfortunately, that's what can happen in a system that has been overwhelmed by the need to provide treatment for many millions of veterans who have been wounded so badly and need constant, ongoing care. Yes, there should be an in-depth investigation and analysis done to get to the bottom of these problems as quickly as possible but the first thing that should be done is to put a gag over the mouths of the many political sharks that suddenly smell blood in the water.
Most Americans know little to nothing about the VA, its good points and bad so let's examine that issue a bit. I'm a member of the VA system but have never used it and don't really intend to unless it's absolutely necessary. Before joining I did some in-depth research about the VA and what I found out was really enlightening and impressive.
It's the largest health care system in America with some 280,000 employees at hundreds of facilities all across the country. It's fully computerized; all hospitals and all members' records are online and available for review by doctors with the proper authority, at any location, at any given time; just like the systems of a national pharmacy. Here's a link that shows how members are classified into 8 specific categories that determine the priority that they receive in being treated.


text:
Next is an album by Soulive that was released on March 12, 2002. It was produced by Jeff Krasno.

Next brought a new chapter into the history of Soulive, and it marked the first time that the band toured as a quartet (with saxophonist Sam Kininger). Next built upon the success of the previous year's release, Doin' Something, with heavy driving beats and harmonized melodies.  Next mirrored the great organ/guitar/sax era of the 60's, and built upon that 60's feel with reminiscent songs like "Tuesday Night's Squad" and "Alkime".  Next also depicted a substantial hip-hop influence with rappers like Black Thought, featured on "Clap!", and Talib Kweli, featured on "Bridge to 'Bama (Hi Tek Remix)".  Dave Matthews was also a guest on the track "Joyful Girl".

Track listing
"Tuesday Night's Squad"  – 7:23
"Flurries"  – 5:55
"Liquid"  – 6:38
"Joyful Girl" (featuring Dave Matthews)  – 6:16
"Kalen"  – 7:39
"Clap!" (featuring Black Thought) – 5:21
"Interlude"  – 1:10
"Ne-Ne"  – 8:06
"I Don't Know" (featuring Amel Larrieux) – 5:09
"Whatever It Is"  – 4:30
"Alkime"  – 7:01
"E.D. Hambone"  – 5:05
"Bridge to 'Bama (Hi Tek Remix)" (featuring Talib Kweli) – 4:36

2002 albums
Blue Note Records albums
Soulive albums

redpajama_set_name: RedPajamaWikipedia


Legends Soccer School recently returned from the Manchester City Cup in Abu Dhabi.
Legends Soccer School, based in Cape Town, recently returned from Abu Dhabi where they took part in the Manchester City Cup.
The tournament, now in its third year, attracts global interest with participating teams from as far as Australia, Sri Lanka, Thailand and the United Kingdom.
The tournament proved to be a unique experience for all involved, and the winning teams from each age group were entered into a draw to win an all-expenses paid trip to the City Football Academy in Manchester.
Organised by Manchester City, the event welcomed teams from the Manchester City Academy, Melbourne City Academy Sri Lanka, Bahrain, and the United Arab Emirates (UAE). Manchester City Academy director Jason Wilcox attended the tournament to watch it unfold.
Legends Soccer School had two teams (u.12, u.14) participate in the tournament.
Both teams performed exceptionally well with the u.12s finishing fifth in their age group, out of 30 teams. The u.14s remained undefeated and reached the final match where they took on Melbourne City Academy, going down 2-0 to bring home the silver medal.
"Without sounding cliché, the experience was truly life-changing. To be invited as the first South African youth team to participate in such a prestigious football event alone is humbling. We all left Cape Town as winners in my eyes," says head coach and director Anees Abbas.
He says their u.12 and u.14 teams worked extremely hard. Legends Soccer School run eight-week programmes in the pre-season from January to March and off-season from October to December. During in-season the school focuses on private (one-on-one) and small group sessions once a week.
"To have been able to take such a diverse group of 22 boys with us on an international trip – of which 16 came from underprivileged backgrounds – will hopefully have an impact in their lives both on and off the field. I am very proud of how they represented South Africa and it is such a joy seeing these young legends take out key lessons from such an experience," he says.
Abbas praised his charges for going toe-to-toe and holding their own with some of the best youth academies in the world.


Terrorism: it's a scary word, and one we'd rather not think about when planning our next trip abroad.
Unfortunately, events like 9/11, the Bali bombings and the attacks in Paris, have altered the way we travel forever. Tighter travel regulations have become the norm and heightened security risks are commonplace.
Whilst these events should not stop travellers from heading abroad, it's simply smart practice to protect your travel plans against the impact of an attack. That being said, plenty of confusion surrounds travel insurance as it relates to terrorism. More specifically, will travel insurance cover an act of terrorism?
To begin with it's important to understand that every travel insurer will handle a terrorist incident differently and will have varying cover exclusions. Although most insurers will generally consider terrorism or acts of war to be an exclusion a few will offer limited cover...keep reading for more.
As soon as a terrorist attack occurs the government will issue travel warnings for that region or country. Restrictions will now apply to those destinations and you would generally not be covered to travel to those regions (particularly those with a level 4 or 5 government warning). Should you choose to travel to a country against government warnings your cover benefits may be rendered void.
In the event of a terrorist attack or political unrest, most if not all policies exclude cover for cancellation fees & lost deposits, disruption of journey or alternative transport expenses. Though it's a difficult pill to swallow, insurers ruled out these specific benefits after the risk of terrorism were deemed too great. However, plenty of insurers will still provide cover for medical cover in the event of a terror attack.
AA Travel Allianz Australia Insurance Limited Covered up to $3,000 for cancellations with the comprehensive cover only.
Certain Underwriters of Lloyds Only covered for cancellation with the Frequent Traveller and Comprehensive Plus plans they allow you to cancel in the event that after you purchase your policy and within a week before you travel, MFAT travel warnings restrict you from travelling.
Your trip must be directly affected by terrorism. You cannot claim for cancellation benefits if your booked accommodation is greater than 2km from where the act of terrorism took place. Only covered for cancellation with the Frequent Traveller and Comprehensive Plus plans they allow you to cancel in the event that after you purchase your policy and within a week before you travel, MFAT travel warnings restrict you from travelling.
World Nomads Certain Underwriters of Lloyds Cover for Kidnap or hijack if you purchase the Explorer Plan.
In the event of a large scale terror attack such as the November Paris attacks, plenty of travel and transport companies responded to customers concerns with humanity, allowing them to postpone their bookings.
However, with the more global threat of terror it can be good practice to have a back-up plan should you need to cancel or delay your trip.
As well, it may be worthwhile looking for accommodation deals that allow free cancellation (often possible on sites like Booking.com and Expedia). Be sure to check the fine print to ensure you're not caught out by any additional fees or upfront pre-payments.
Flexible fares may be more expensive but could pay off when weighed up against the potential emotional and financial cost of a trip cancellation. It may also be worth looking into package holidays. Although these won't neccessarily have flexible cancellation policies, the operator will be under legal obligation to ensure your safety in a crisis.
A few insurers do provide cover for medical and hospital expenses caused by an act of terrorism, including the cost of bringing you back home. Keep reading to see who covers what. If your insurer is not on the list, it's crucial to review your policy coverage, as every insurer will vary in their benefits.
In any case, medical cover is essential for travellers headed abroad. The risk of being exposed to illness or injury is simply not one worth taking once you consider the full cost of medical and hospital expenses overseas. Add to that the expense of medical evacuation, accidental death or total permanent disability and the need for travel insurance cannot be over emphasised.
Whilst you not be able to rely on travel insurance for all terrorism related claims, it is still vital to take out a policy before you head away. The New Zealand's Ministry of Foreign Affairs and Trade(MFAT) website, SafeTravel can be a very useful resource and one worth checking regularly to stay abreast of any current events. The New Zealand government can help travellers during and after a terrorist incident, so be sure to register your travels with SafeTravel as well.
Although travelling in times of uncertainty can be nerve-wracking, it can be helpful to take some perspective. Statistically speaking, the chances of being impacted by an act of terrorism abroad are minimal. However, when it comes to particularly dangerous destinations, it is worth considering your need to travel. Always take travel insurance along with you and keep an eye on current events. For more information on safe travel check out our guide to travel warnings.
Can no longer go on your trip?
View our cancellation cover guide.
What is covered should a family emergency occur?


text:
REVIEW: O-Antigen Modifications Providing Antigenic Diversity of Shigella flexneri and Underlying Genetic Mechanisms
Y. A. Knirel1*, Qiangzheng Sun2, S. N. Senchenkova1, A. V. Perepelov1, A. S. Shashkov1, and Jianguo Xu2
1Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia; fax: (499) 137-6148; E-mail: yknirel@gmail.com
2State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing 102206, China; E-mail: sunqiangzheng@icdc.cn; xujianguo@icdc.cn
Received December 11, 2014; Revision received February 24, 2015
O-Antigens (O-specific polysaccharides) of Shigella flexneri, a primary cause of shigellosis, are distinguished by a wide diversity of chemical modifications following the oligosaccharide O-unit assembly. The present review is devoted to structural, serological, and genetic aspects of these modifications, including O-acetylation and phosphorylation with phosphoethanolamine that have been identified recently. The modifications confer the host with specific immunodeterminants (O-factors or O-antigen epitopes), which accounts for the antigenic diversity of S. flexneri considered as a virulence factor of the pathogen. Totally, 30 O-antigen variants have been recognized in these bacteria, the corresponding O-factors characterized using specific antibodies, and a significant extension of the serotyping scheme of S. flexneri on this basis is suggested. Multiple genes responsible for the O-antigen modifications and the resultant serotype conversions of S. flexneri have been identified. The genetic mechanisms of the O-antigen diversification by acquisition of mobile genetic elements, including prophages and plasmids, followed occasionally by gene mobilization and inactivation have been revealed. These findings further our understanding of the genetics and antigenicity of S. flexneri and assist control of shigellosis.
KEY WORDS: Shigella flexneri, O-antigen, O-polysaccharide structure, serotype-converting bacteriophage, transposon, plasmid, serotyping, immunodeterminant
Abbreviations: GalA, galacturonic acid; GalNAc, 2-acetamido-2-deoxygalactose; GlcNAc, 2-acetamido-2-deoxyglucose; IS, insertion sequence; LPS, lipopolysaccharide; PEtN, phosphoethanolamine; Rha, rhamnose; Sf, Shigella flexneri.
Shigellosis, or bacillary dysentery, is an acute diarrheal disease that remains an important public health challenge, especially in developing countries. There are an estimated about 164.7 million shigellosis cases annually worldwide causing 1.1 million deaths, with the majority involving children under five years old [1]. The causative agent of shigellosis is Shigella spp., nonmotile, nonspore-forming facultative anaerobic Gram-negative bacteria, which are among the bacterial pathogens most frequently isolated from patients with diarrhea. Invasion by these bacteria of the colonic and rectal mucosa and the following inflammatory response provoke massive mucosal destruction reflected in strong abdominal cramps and stools containing blood and mucus [2]. Based on biochemical properties and O-antigen specificity, the genus Shigella is divided into four species or subgroups, including S. dysenteriae, S. flexneri, S. boydii, and S. sonnei [3], although genetically they all but S. boydii type 13 are clones of Escherichia coli [4]. Shigella flexneri is the predominant species causing shigellosis in developing countries and the second, after S. sonnei, most common in industrialized countries [5-7].
Being a serologically heterogeneous species, S. flexneri is further divided into various serotypes and subtypes. A commercially available monovalent antisera kit (Denka Seiken, Japan) and monoclonal antibody reagents (MASFs) (Reagensia AB, Sweden) are widely used for serotyping S. flexneri isolates. The serospecificity of S. flexneri is defined by a combination of immunodeterminants (O-factors), which reside on the O-antigen located on the bacterial cell surface [3]. The O-antigen, also called O-specific polysaccharide or O-polysaccharide, is a part of the outer-membrane lipopolysaccharide (LPS) and is linked to the lipid moiety (lipid A) via a core oligosaccharide. The O-polysaccharide consists of many oligosaccharide repeats (O-units). Structure and serology of the O-antigens of S. flexneri have been intensively studied for the last 50 years ([8-10] and references therein).
The O-antigens of S. flexneri are synthesized by the O-antigen polymerase (Wzy)/flippase (Wzx)-dependent pathway, whereby the O-unit is preassembled on a lipid carrier on the cytoplasmic side of the inner membrane, and after translocation (flipping) to the periplasmic side mediated by Wzx, is polymerized by Wzy with participation of a chain length regulator Wzz. There are two basal S. flexneri O-polysaccharide structures and, correspondingly, two non-variable gene clusters for O-antigen biosynthesis: one for serotype 6 and the other for the remaining serotypes. The bacteria of the two groups have different evolutionary origins and belong to different lineages of Shigella clones of E. coli [4]. As in most E. coli clones including the other Shigella spp., the O-antigen gene cluster maps between the housekeeping genes galF and gnd on the chromosome [11]. It contains genes for synthesis of a nucleotide (deoxythymidine diphosphate) precursor of l-rhamnose, a specific monosaccharide component of both basal O-polysaccharides, for glycosyltransferases necessary for the assembly of the O-unit, and O-antigen processing genes: wzx for flippase and wzy for O-antigen polymerase.
Molecular typing targeting specific genes in the O-antigen cluster, including wzx, wzy, and glycosyltransferases genes, detects all S. flexneri non-6 serotypes as a single group [12]. Escherichia coli O13, O129, and O135 having the same basal O-antigen structure [10, 13] and essentially identical O-antigen gene cluster [11] also fall in this group, and, similarly, E. coli O147 forms one molecular group and shares the O-antigen structure with S. flexneri serotype 6 [11, 12]. The closely related E. coli and S. flexneri clones can be differentiated using PCR assays based on other genes not related to the O-antigen synthesis [12].
The O-antigens of S. flexneri non-6 serotypes are highly diverse due to various chemical modifications to the basal structure giving rise to the observed serological heterogeneity [3]. A number of genes outside the O-antigen cluster are involved in the modifications [14], which occur after the O-unit assembly and before the transfer of the mature O-polysaccharide to the lipid A-core region of the LPS. A molecular approach targeting specific O-antigen modification genes identified by that time has been developed for serotyping S. flexneri within the group of non-6 serotypes [15].
The O-antigen plays an important role in the pathogenesis of S. flexneri; particularly, it protects the bacteria from the lytic action of serum complement and promotes adherence and internalization of bacteria to intestinal epithelial cells [16-18]. Creating antigenic diversity by O-antigen modifications is considered as an important virulence factor of S. flexneri that enhances survival of the pathogens because the host has to mount a specific immune response to each serotype [19]. Moreover, such modification as glucosylation at certain sites promotes invasion of S. flexneri into host cells mediated by the type III secretion system [16].
In previous reviews devoted to S. flexneri O-antigens [9, 14, 19], modifications known by that time, including glucosylation at various sites and O-acetylation at one site (on RhaI), have been considered in detail. Recently, more sites of O-acetylation [10, 13, 20-25] and a novel modification type, phosphorylation with phosphoethanolamine (PEtN) [26-29], have been identified and genetic bases of the new modifications have been elucidated. The present review summarizes structural, serological, and genetic aspects of the O-antigen modifications in S. flexneri with emphasis on the new findings.
STRUCTURES AND IMMUNOSPECIFICITY OF THE O-ANTIGENS
Except for serotype 6, all known S. flexneri serotypes (1-5, 7, X, Y) share the O-polysaccharide backbone (1) composed of tetrasaccharide O-units containing three l-rhamnose residues (RhaI-RhaIII) and one residue of 2-acetamido-2-deoxy-d-glucose (GlcNAc) [30].
→2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→4)-α-l-RhapI-(1→3)-β-d-GlcpNAc-(1→ (1)
The polysaccharide (1) present in serotype Y is characterized by two antigenic specificities labeled dual group O-factor 3,4. A structural domain that defines this O-factor has not been completely identified yet [31, 32]. In some cases, its manifestation is ambiguous as strains otherwise identical in the O-antigen structure and the presence of other immunodeterminants may express or may not express O-factor 3,4 (e.g. former serotypes 3b and 3c, which have been proposed to be combined into one serotype 3b [10]). The polysaccharide (1) can be modified by adding various chemical groups (α-d-glucopyranosyl, O-acetyl, phosphoethanolamine) to different sugars giving rise to enormously diverse O-antigen structures and, correspondingly, to serological heterogeneity, which is the basis for serotyping of S. flexneri strains (table).
Structures of the O-polysaccharides of S. flexneri. Included are both serotypes already approved internationally and provisional serotypes that express epitopes associated with newly identified O-acetyl and PEtN groups
Note: RhaIII and GlcNAc are O-acetylated non-stoichiometrically. A minor 4-O-acetylation on RhaIII that occurs alternatively to the major 3-O-acetylation on RhaIII is not shown. In the antigenic formulae, type and group O-factors are indicated before and after colon, respectively. O-factor 3,4 associated with the O-polysaccharide backbone is variably expressed and, except for serotype Y, is omitted from the antigenic formulae in the table.
a-e Authors' unpublished data.
a Strain 2005122; the degree of 6-O-acetylation on GlcNAc ~75%.
b Strain 2001019.
c Strain 2005128; the degree of 6-O-acetylation on GlcNAc ~75%.
d Strain 06AH74; the degree of 3/4-O-acetylation on RhaIII ~40/25%.
e Strains 06HN054 and 06HN303.
f Proposed based on the reactivity with MASF IV-1 [45, 46] with no data on the O-antigen structure and genetics available.
Glucosylation may occur on any monosaccharide in the polysaccharide (1) giving rise to type O-factors I, II, IV, and V when present at various positions on RhaI, RhaII, or GlcNAc, and to dual group O-factor 7,8 when present on RhaIII (Fig. 1). The type O-factors define serotypes 1, 2, 4, and 5, respectively, whereas the group O-factor 7,8 may be expressed in different serotypes and occur in combination with various type O-factors [8, 9, 34, 35] (table). As a result, in the O-units of some subtypes there are two side-chain glucosyl groups. The degree of glucosylation at each position is close to stoichiometric, but the first O-unit of the O-polysaccharide chain linked to the LPS core lacks any glucosyl residue [39-41].
Fig. 1. Immunodeterminants (O-factors) associated with various O-antigen-modifying groups.
In serotype 7, GlcNAc carries the α-d-Glcp-(1→2)-α-d-Glcp-(1→ disaccharide [37, 38], which defines O-factor VII (IC). Subtype 7a was originally called 1c [37], but wild-type strains of this subtype do not react with antibodies against O-factor I. Therefore, it was suggested to rename it 7a and to replace the type O-factor IC with VII in the serotyping scheme of S. flexneri [38].
O-Acetylation has been identified on RhaI, RhaIII, and GlcNAc [10, 22-24, 33, 38, 42] (Fig. 1). The degree of O-acetylation is variable and depends likely not only on strain but also on storage and cultivation conditions. On RhaI, the 2-O-acetylation is stoichiometric or close to stoichiometric, whereas on GlcNAc, 6-O-acetylation varies from 30 to 75%. RhaIII is O-acetylated at position 3 in some O-units and at position 4 in some others (3/4-O-acetylation), the former being the major (25-70%) and the latter the minor (15-25%) modification site. All combinations of O-acetylated and non-acetylated RhaIII and GlcNAc have been found in the O-units of the serotype 2a O-polysaccharide, and hence O-acetylation on both residues is random [20]. In a short-chain LPS having a single O-unit, GlcNAc lacks 6-O-acetylation and RhaIII is mono-O-acetylated at any position [40].
O-Acetylation on RhaI, RhaIII, and GlcNAc defines group O-factors 6, 9, and 10, respectively. Serotype 3 strains express type O-factor III, which depends on the same 2-O-acetyl group on RhaI as group O-factor 6 [42] but, in contrast to the latter, is abolished by glucosylation on GlcNAc in serotypes 1b, 4b, and 7b. O-Factor III could be recovered from serotypes 1b and 4b by transformation with the functional oacD gene, which resulted in partial 6-O-acetylation (~25-30%) accompanied by deglucosylation on GlcNAc [24]. 6-O-Acetylation on GlcNAc also occurs in the common enterobacterial polysaccharide antigen [43, 44] and, as a result, O-factor 10 is expressed by some other enteric bacteria, including Shigella sonnei phase II [24].
In early studies of S. flexneri O-antigen structures, O-acetylation on RhaIII and GlcNAc has been overlooked, and the corresponding O-factors 9 and 10 have not been included in the S. flexneri serotyping scheme. To fill the gap, we suggest to further divide the existent serotypes into O-factor 9- and 10-positive and -negative subtypes (i) by keeping the old names for the subtypes that lack both O-factors 9 and 10 [24, 33] and for serotype 1b whose O-factor 9-negative variant has not been found in nature, and (ii) by indication of expression of one or both of the O-factors 9 and 10, by adding subscript 1 or 2, respectively (e.g. 2a1 and 2a2 for subtypes characterized by the antigenic formulae II: 10 and II: 9; 10, respectively). To distinguish these subtypes, monospecific antisera against O-factors 9 [33] and 10 [24] that have already been generated and verified should be included into the serological diagnostic kit.
Phosphorylation with PEtN has been reported in subtypes 4av, Xv, Yv, and Yv1 designated as "variant" subtypes by adding letter "v" to the names of the corresponding PEtN-positive subtypes [26-29]. Stoichiometric phosphorylation occurs at position 3 of RhaIII in subtype 4av [26] or RhaII in subtype Xv [27] (Fig. 1) with minor phosphorylation (~10%) on the neighboring rhamnose residue, in subtype Xv the minor PEtN group replacing the glucosyl group on RhaIII [27]. In subtypes Yv and Yv1, both rhamnose residues are phosphorylated, one being completely (RhaII in subtype Yv or RhaIII in subtype Yv1) and the other partially modified ([28] and authors' unpublished data). It has been demonstrated that in subtype Yv1, bisphosphorylation occurs in the non-O-acetylated O-units only, and the Yv1 O-polysaccharide is composed of blocks of repeats differing in the number of PEtN groups and the presence or absence of O-acetylation [28].
O-Acetylation on RhaIII and GlcNAc as well as phosphorylation with PEtN has not been identified in early structural studies of S. flexneri O-antigens, but monoclonal antibody MASF IV-1 generated against a PEtN-positive strain of subtype 4a (now 4av) has been included into the MASF reagents (Reagensia AB). This antibody is useful for detection of all PEtN-positive strains, as the corresponding group O-factor IV-1 (originally called 4X [31]) is defined by phosphorylation with PEtN whether it occurs on RhaII or RhaIII [27, 29]. That the same monoclonal antibody recognizes a PEtN-associated epitope on either of the two monosaccharides is probably due to a sharp turn of the polysaccharide chain at each 2-substituted Rha residue, which makes both RhaII and RhaIII easily accessible to interaction with the protein and neglects the role of the neighboring sugar residues.
The serotype 6 O-polysaccharide (2) is acidic due to the presence of d-galacturonic acid (GalA) [10, 36]. The first monosaccharide in the serotype 6 O-unit is 2-acetamido-2-deoxy-d-galactose (GalNAc) rather than GlcNAc, but the α1→2-linked rhamnose disaccharide at the other side of the O-unit is shared by all S. flexneri serotypes.
→2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→4)-β-d-GalpA-(1→3)-β-d-GalpNAc-(1→ (2)
Serotype 6 strains are recognized by typing antiserum VI specific to an unidentified O-polysaccharide domain. The only modification of the serotype 6 O-antigen that has been characterized chemically is 3/4-O-acetylation on RhaIII [10, 33]. Due to the presence of an O-polysaccharide backbone fragment in common with non-6 serotypes of S. flexneri, this modification confers serotype 6 with O-factor 9, which is recognized smoothly by antiserum 9 produced against an O-factor 9-positive strain of serotype 2 [23, 33]. O-Factor 9 is present in all serotype 6 strains tested [10, 23, 33]. As in serotype 2a, the terminal RhaIII residue of the single O-unit in a short-chain serotype 6 LPS is O-acetylated randomly [40].
Seven atypical serotype 6 strains collected in Bangladesh during 1985-1987 [45] and 1997-2000 [46] were recognized by monoclonal antibody MASF IV-1, suggesting that subtype 6v carrying a PEtN-associated epitope emerged in nature. Structural and genetic bases of the O-factor IV-1 expression in this subtype remain to be elucidated.
GENETIC BASIS OF O-ANTIGEN MODIFICATIONS AND RESULTANT SEROTYPE CONVERSION
Glucosylation. Three Gtr proteins (GtrA, GtrB, and type-specific Gtr (Gtr(type)) mediate glucosylation of the O-polysaccharide backbone (1). GtrA and GtrB are highly conserved and functionally interchangeable between serotypes. GtrB catalyzes synthesis of undecaprenyl phosphate-β-glucose (UndP-β-Glc) from UDP-α-Glc, and GtrA functions as flippase allowing translocation of the UndP-β-Glc from cytoplasm to periplasm. The third protein, Gtr(type), is a serotype-specific glucosyltransferase (GtrI, GtrII, GtrIV, GtrV, GtrVII (formerly GtrIc), and GtrX) responsible for the transfer of the glucosyl group from UndP-β-Glc to a certain position of one of the sugar residues of the growing O-polysaccharide chain. The Gtr(type) enzymes are integral membrane proteins consisting of 8-10 transmembrane helices with the active sites located in the large periplasmic loops at the N- and C-termini. They have weak similarity to other known glucosyltransferase families and are predicted to be members of the GT-C superfamily, which utilize a phospholipid-activated donor sugar substrate [47].
A single operon on the chromosome encoding Gtr proteins (gtr cluster) is carried by a (cryptic) prophage acquired by lysogeny of the bacteria with one or two from five temperate bacteriophages (SfI, SfII, SfIV, SfV, and SfX) [14, 48]. Each prophage (or a couple of prophages in the case of bisglucosylation in the O-unit) is integrated into the thrW tRNA gene in the same region adjacent to the proA gene in the S. flexneri genome. The gtr cluster is localized immediately downstream of the phage attachment site attP, which follows the integrase (int) and excisionase (xis) genes. All bacteriophages have been isolated from the corresponding S. flexneri strains and well characterized [41, 46-54].
Lysogeny with bacteriophages SfI, SfII, SfIV, SfV, and SfX converts serotype Y to serotypes 1a, 2a, 4a, 5a, and X, respectively (Fig. 2), whereas the potential recipient range among other serotypes is quite different. This is single serotype X for SfI, two serotypes (3b and 5a) for SfII, two serotypes (2a1 and 3b) for SfX, four subtypes of serotypes 1a, VII, and X for SfIV, and six subtypes of serotypes 1-4 for SfV of the 12 serotypes tested. The limitation in the host recognition is evidently due to the phage immunity from a modified O-antigen, which constitutes the receptor for the phage adsorption on the cell surface, a mechanism by which lysogeny prevents subsequent infection of bacteria by homologous or related phages ([52-54], Q. Sun, J. Wang, X. Luo et al., unpublished data). Accordingly, the order of lysogeny with two phages giving rise to serotypes carrying more than one phage-borne modification factor can be constrained; for instance, phage SfI can infect SfX-carrying serotype X strains giving rise to serotype 1d, but serotype 1 strains are resistant to phage SfX [55] (Fig. 2).
Fig. 2. Conversion pathways of serotypes 1-5, 7, X, and Y mediated by bacteriophages SfI, SfII, SfIV, SfV, SfVII, SfX, Sf6 or Sf61b, Sf101, and plasmids pSFxv_2 or pSFyv_2. Adopted from ([24, 25, 27, 29, 52-56, 59], Q. Sun, J. Wang, X. Luo et al., unpublished data). Antigenic formulae are shown in parentheses (except for serotype Y, group O-factor 3,4 associated with the O-polysaccharide backbone is omitted). Asterisk indicates gene inactivation. Dashed arrows show that the order of serotype-converting events is unknown. Putative intermediates that have not been found in nature are shown in dashed rectangular box. Hypothetical bacteriophages Sf61b and SfVII have not been isolated. The bacteriophage Sf101 origin of the oacB gene has been demonstrated for two serotype 7a1 strains, and mobilization of oacB into other 3/4-O-acetylation-carrying 7a and non-7a strains suggested to result from disruption of the Sf101 prophage by IS (insertion sequence) elements followed by recombination [25].
Inactivating mutations in the gtr locus occur in a number of wild-type strains that carry a serotype-converting phage, resulting in their reversion to the parental serotype (Y) or (in case of bisglucosylation) an intermediate serotype (Fig. 2). For instance, from 35 serotype Y strains, 13 strains possess defective gene gtrII and six strains defective gene gtrI. From 19 strains of O-factor IV-1-positive serotype Yv and Yv1, 13 strains have mutations in either one or both genes gtrII and gtrB, and three strains possess a defective gtrX [29]. As a result, the same serotype may have multiple origins, e.g. subtypes Yv and Yv1 emerged independently at least three times from serotypes Y, Xv, and 2a by acquisition of an opt-carrying plasmid, inactivation of a gtr gene, and both events, respectively [29] (Fig. 2).
In serotype 7 that is distinguished by the presence of the side-chain α1→2-linked glucose disaccharide, the addition of the first glucosyl group is mediated by the same gtr cluster within a SfI prophage as in serotype 1 [56]. The gtrVII gene designated originally gtrIC codes for the serotype 7-specific glucosyltransferase that mediates addition of the second glucose residue to the first one. As the other type-specific gtr(type) genes, gtrVII is present as part of a three-gene gtr cluster but is located at a different place on the chromosome adjacent to the conserved yejO locus. It is distantly related to the other S. flexneri gtr clusters and appears to have been acquired from outside the species, presumably via infection by a hypothetical bacteriophage SfVII (SfIC) [56].
O-Acetylation. 2-O-Acetylation of RhaI is mediated by an acetyltransferase, which was originally named Oac, but after discovery of other O-antigen-modifying acetyltransferases in S. flexneri, it was suggested to rename it OacA [22]. The receptor for OacA is the O-antigen of serotype Y having the basal structure (1) as well as some other serotypes (Fig. 2). OacA consists of 10 α-helical membrane-spanning regions with both the N- and C-termini located in the cytoplasm. It bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane regions [57]. In serotypes 3a, 3b, and 4b, the oacA gene and an adjacent integrase-encoding gene are carried by the temperate bacteriophage Sf6, which, like the gtr locus-carrying bacteriophages, is a member of the canonical lambdoid phage group. The Sf6 genome is integrated into the argW tRNA gene of the host chromosome next to the conserved yfdC gene (Fig. 3a) [58].
Fig. 3. Organizations of the genomic regions of bacteriophages and S. flexneri carrying the O-acetyltransferase genes: oacA (former oac) in bacteriophage Sf6 and serotypes 3a (subtype 3a1), 3b, 4b, and oacA1b (former oac1b) in serotypes 1b and 7b (a); oacD in bacteriophage SfII and serotype 2a (subtype 2a2, strain Sf301), oacB in bacteriophage Sf101, serotype 7a (subtype 7a1, strain SFL1683) and 2a (b); oacC in serotype 6 (strains CCH 060 (top) and CDC 796-83 (bottom)) (c). Adopted from [22-25, 57, 59]. The gtr locus for type II glucosylation, the O-acetyltransferase genes, and the conserved flanking genes are shown in light gray, dark gray, and black, respectively. attP indicates attachment site on phage, attL and attR indicate left and right ends of the integrated phage genome.
Serotypes 1b [59] and 7b (authors' unpublished data) possess a variant oacA gene named oacA1b (originally oac1b), which shares with oacA 88-89% identity at the DNA level and 85% identity at the protein level. Despite the rather high sequence variation, oacA and oacA1b are functionally interchangeable in 2-O-acetylation of RhaI. oacA1b is located in a chromosomal region between the conserved torT and ycmA genes, which evidently has a phage origin but is different from the Sf6 phage genome (Fig. 3a). Whereas serotypes 3a, 3b, and 4b can be generated by infecting with bacteriophage Sf6 strains of serotypes X, Y, and 4a, respectively, this phage cannot convert serotype 1a into serotype 1b. Therefore, it is likely that oacA1b has been obtained from outside S. flexneri, probably by infection with another bacteriophage (hypothetical Sf61b) rather than evolved by divergence from the oacA gene.
6-O-Acetylation of GlcNAc is mediated by an oac homolog designated oacD, which is carried by SfII bacteriophage also responsible for 4-glucosylation of RhaI giving rise to serotype 2 (Fig. 3b) [24]. The occurrence of an insertion sequence (IS) upstream of oacD suggests that this gene was incorporated into the SfII genome by an insertion event. The functional oacD gene also is present in strains of several non-2 serotypes (3a1, X1, Y1, Y2, and Yv1) that carry a cryptic SfII prophage with a dysfunctional gtr locus for the type II glucosylation. Therefore, the OacD-mediated 6-O-acetylation of GlcNAc does not depend on the functional gtr locus.
3/4-O-Acetylation of RhaIII that occurs in subtypes 1a1, 1b, 2a2, 5a1, 71, Y1, 6, and Y2 [22, 25] is mediated by another Oac homolog called OacB. This modification was unaffected by transformation of 2a2 and Y1 strains with either the gtrABX locus for 3-O-glucosylation (authors' unpublished data) or the optII gene for 3-O-phosphorylation [60] of RhaIII. In contrast, transformation of 2b and X strains with oacB from a 2a2 strain resulted in their conversion into serotypes 2a and Y, respectively, due to replacement of 3-O-glucosylation with 3/4-O-acetylation on RhaIII [22]. The mechanism that makes the O-acetylation the preferable modification on RhaIII remains to be elucidated.
Two alternative locations of the oacB gene on the bacterial chromosome have been reported. In some subtype 7a1 strains, oacB is carried by Sf101 prophage integrated in the sbcB gene next to yeeD [22] (Fig. 3b). In several different subtype 7a1 strains [25] and 3/4-O-acetylation-carrying strains of other serotypes [22], oacB maps upstream of the adrA gene in the same proA-adrA region on the chromosome, in which the gtr-carrying prophages are integrated. It is located downstream of an integrase-encoding gene (int), and the int-oacB locus is flanked by IS elements giving rise to a transposon-like structure [22]. In serotype 2a strains examined, this structure is located immediately downstream of the SfII prophage genome (Fig. 3b).
In serotype 6, yet another oac homolog, oacC, common for all strains of this serotype, is responsible for the 3/4-O-acetylation of RhaIII (Fig. 3c) [23]. It maps in a phage-like structure localized in yet another place on the chromosome (Fig. 3c). OacB and OacC have high sequence homology (72% identity) and interchangeable function in mediating the 3/4-O-acetylation of RhaIII. This is not surprising as the O-polysaccharides of all S. flexneri serotypes share a →2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→ disaccharide fragment, which evidently serves as the acceptor substrate for both OacB and OacC. The three known rhamnose-modifying acyltransferases OacA-OacC present higher homology in the regions conserved among the inner membrane trans-acylase family proteins [23]; particularly, conserved are amino acid residues R73 and R76, which are known to be critical for Oac functioning [57]. The divergent oac genes might have been gained from different bacterial species in independent events.
Phosphorylation with PEtN. A polymorphic opt gene (originally called lpt-O) encoding for PEtN transferases is responsible for adding a PEtN group to RhaII or/and RhaIII in subtypes 4av, Xv, Yv, and Yv1 [27, 28]. The Opt proteins have been predicted to belong to the sulfatase superfamily and to contain a sulfatase domain on the carboxyl terminus, which putatively is involved in catalyzing the transfer of PEtN to a sugar residue. Two functionally interchangeable opt alleles, optII and optIII, are borne by double-stranded circular plasmids 6850 bp in length called pSFxv_2 and pSFyv_2, respectively (Fig. 4). OptII and OptIII preferentially mediate phosphorylation of RhaII and RhaIII [27-29], and, accordingly, optII and optIII are present in subtypes Xv and 4av, respectively. An explanation for the evolution of this gene may be selection pressure as in serotype 4a, RhaIII is not occupied, and the OptIII can easily mediate the addition of PEtN onto it. In contrast, in serotype X, RhaIII carries a glucosyl group, and the OptIII-mediated phosphorylation may not effectively compete for RhaIII with type 7,8 glucosylation, whereas OptII can smoothly modify RhaII. In subtypes Yv and Yv1, the opt form depends on the strain origin: this is optII in Yv strains derived from serotype Y or Xv or optIII in serotype 2-derived Yv1 strains (Fig. 2) [29]. Accordingly, RhaII and RhaIII are predominantly phosphorylated in subtypes Yv and Yv1, respectively ([28] and authors' unpublished data).
Fig. 4. Genomic structure of plasmid pSFxv_2 carrying the PEtN transferase gene optII. Adopted from [27]. repA, replication initiation protein gene; hp, hypothetical protein.
It has been demonstrated that plasmids pSFxv_2 [60] and pSFyv_2 (authors' unpublished data) can be transferred into, and stably maintained in, strains of other S. flexneri serotypes (1 to 6) giving rise to unnatural O-factor IV-1-positive serovariants. Upon the transformation, the initial serospecificity is either retained or lost, or manifestation of an initial epitope(s) may be weakened. Phosphorylation with PEtN may interfere with other modifications on the O-antigen not only on the same monosaccharide but also on different sugar residues; for instance, 3-O-phosphorylation on RhaII is incompatible with 4-O-glucosylation on RhaI [60]. This phenomenon seems to account for the absence of the PEtN-carrying variant of 2a (subtype 2av) in nature, while its non-phosphorylated form is highly prevalent among clinical isolates. It also shows that in the emergence of subtype Yv1 from subtype 2a1, inactivation of gtrII or gtrB and the resultant loss of type II glucosylation occurred first and pSFyv_2 plasmid was gained subsequently (Fig. 2).
Known modifications of the O-antigens of S. flexneri involve glucosylation, O-acetylation, or/and phosphorylation with PEtN at various monosaccharides in the O-unit. None is unique on its own, but their most diverse combinations provide an unusually high diversity of the O-antigen forms. By now, about 30 structural variants of the O-antigens of S. flexneri with the same basal structure have been identified, which exceed significantly their number in any other bacteria.
Glucosylation has been known to take place at the periplasmic side of the inner membrane together with the O-unit polymerization [14]. The data on the O-acetylation summarized earlier [14] are contradictory, and it remains unknown where phosphorylation of the O-antigen takes place. Some modifications on different monosaccharide residues, e.g. 4-O-glucosylation on RhaI and 3-O-phosphorylation on RhaII, are incompatible with each other. The modification pattern of the first O-unit that is linked to the LPS core may be different; particularly, glucosylation only happens to at least the second O-unit from the growing end, the first one being unaltered [40]. This finding and the fact that the same Wzy polymerase catalyzes synthesis of structurally different O-polysaccharides indicate complex interactions of the enzymes involved in the O-antigen polymerization and modifications, which remain to be elucidated.
The diverse O-polysaccharide modifications are encoded by a number of genes outside the O-antigen clusters between galF and gnd, and their roles have been elucidated by serological analysis of the wild-type and non-polar mutants and structure determination of their isolated O-polysaccharides. Multiple genetic mechanisms have been recognized to underlie the S. flexneri O-antigen modifications and the resultant serotype conversions.
Most common is lysogeny by bacteriophages that encode glucosyltransferases and/or acetyltransferases. One of the bacteriophages, SfII, includes both gtr gene cluster for 4-O-glucosylation of RhaI and oacD gene for 6-O-acetylation of GlcNAc [24]. This is a unique situation in S. flexneri that one serotype-converting phage carries two genetic factors involved in different types of O-antigen modifications.
Another way of O-antigen modification factor mobilization is a combination of the oacB gene for 3/4-O-acetylation of RhaIII with several IS elements giving rise to a transposon-like structure [22], which could evolve as a result of disruption of oacB-carrying Sf101 prophage by IS elements [25].
Most gtr-containing prophages and the oacB-carrying locus map in the same region on the chromosome upstream of the adrA gene, which seems to be a conserved insertion site for mobile genetic elements. On the other hand, the gtr locus for addition of the second glucose residue in serotype 7 as well as oacB in some subtype 7a1 strains and genes for all other acetyltransferases are parts of bacteriophage genomes or phage-like structures integrated at different places on the chromosome. The polymorphic opt gene that encodes for PEtN-transferases responsible for phosphorylation of RhaIII or/and RhaII, is carried on 6.85-kb plasmids, which have high dissemination potential among S. flexneri serotypes ([60] and authors' unpublished data).
Therefore, the primary genetic mechanism of diversification of S. flexneri non-serotype 6 O-antigen structures is acquisition of various transferable genetic factors, including prophages and plasmids, which can easily spread among different strains. Inactivation of a gene involved in an O-antigen modification, such as a gtr gene, oac, or opt, contributes to further conversion of serotypes. Because of the involvement of multiple O-antigen modification factors, the same serotype may emerge multiple times not only by the same [61] but also by different ways, including both gaining and losing one or several factors (Fig. 2).
Recent identification of PEtN phosphorylation and O-acetylation at new sites has refined the notion of the antigenic heterogeneity of S. flexneri (table and Fig. 2). It has become clear that the diversity of the O-antigenic forms in these bacteria was underestimated in the past. For their separate detection, it was proposed to apply specific antisera obtained by absorption of immune sera against wild-type strains with the corresponding isogenic mutants or vice versa [23, 24, 27, 28, 33, 62, 63], as well as molecular approaches using as targets the specific genes responsible for the O-antigen modifications [22-24, 27, 28, 61]. Serological and molecular screening showed that most newly found O-antigen forms occur rather frequently. The data show the expediency of an extension of the existing S. flexneri serotyping scheme by recognition of the representative strains of the new variants as distinctive subtypes.
Human immune response to S. flexneri infection is serotype specific with protection against subsequent infection by the same serotype only. As acquisition of multiple drug resistance, the appearance of new surface epitopes due to O-antigen modifications would be expected to offer a significant advantage to the pathogen and to promote its spread in human populations. For instance, serotype Xv distinguished by PEtN phosphorylation of RhaII appeared initially in one province in China in 2001 and rapidly expanded to most provinces, surpassing 2a as the predominant serotype [62]. It has been demonstrated that glucosylation on RhaI, RhaII, and GlcNAc confers a specific advantage on S. flexneri [16], and it may be suggested that 3/4-O-acetylation on RhaIII is somehow beneficial to the bacteria too, as it occurs in >95% strains of serotypes 1a, 1b, and 2a, which are predominant in developing countries.
Therefore, the data presented in this review, including elucidation of the finer details of the O-antigen modifications and the underlying genetic mechanisms, shed light, and provide avenues for further studies, on the role of the O-antigen variations in the antigenicity, pathogenicity, and epidemicity of S. flexneri. They also have profound implications in development of improved diagnostic methods and efficient shigellosis vaccines targeting newly discovered genes and epitopes.
We acknowledge our colleagues who have significantly contributed to the advancement of this field.
This work was supported by the Russian Science Foundation (Grant No. 14-14-01042 for Y. A. K.). Q. S. and J. X. were supported by the National Natural Science Foundation of China (No. 81271788, 81290340 and 81290345) and the National Key Program for Infectious Diseases of China (2013ZX10004221, 2013ZX10004216-001-002).
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13.Perepelov, A. V., Shevelev, S. D., Liu, B., Senchenkova, S. N., Shashkov, A. S., Feng, L., Knirel, Y. A., and Wang, L. (2010) Structures of the O-antigens of Escherichia coli O13, O129 and O135 related to the O-antigens of Shigella flexneri, Carbohydr. Res., 345, 1594-1599.
14.Allison, G. E., and Verma, N. K. (2000) Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri, Trends Microbiol., 8, 17-23.
15.Sun, Q., Lan, R., Wang, Y., Zhao, A., Zhang, S., Wang, J., Xia, S., Jin, D., Cui, Z., Zhao, H., Li, Z., Ye, C., Jing, H., and Xu, J. (2011) Development of a multiplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella flexneri, J. Clin. Microbiol., 49, 3766-3770.
16.West, N. P., Sansonetti, P., Mounier, J., Exley, R. M., Parsot, C., Guadagnini, S., Prevost, M. C., Prochnicka-Chalufour, A., Delepierre, M., Tanguy, M., and Tang, C. M. (2005) Optimization of virulence functions through glucosylation of Shigella LPS, Science, 307, 1313-1317.
17.Kohler, H., Rodrigues, S. P., and McCormick, B. A. (2002) Shigella flexneri interactions with the basolateral membrane domain of polarized model intestinal epithelium: role of lipopolysaccharide in cell invasion and in activation of the mitogen-activated protein kinase ERK, Infect. Immun., 70, 1150-1158.
18.Hong, M., and Payne, S. M. (1997) Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance, Mol. Microbiol., 24, 779-791.
19.Brahmbhatt, H. N., Lindberg, A. A., and Timmis, K. N. (1992) Shigella lipopolysaccharide: structure, genetics, and vaccine development, Curr. Top. Microbiol. Immunol., 180, 45-64.
20.Kubler-Kielb, J., Vinogradov, E., Chu, C., and Schneerson, R. (2007) O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a, Carbohydr. Res., 342, 643-647.
21.Perepelov, A. V., L'vov, V. L., Liu, B., Senchenkova, S. N., Shekht, M. E., Shashkov, A. S., Feng, L., Aparin, P. G., Wang, L., and Knirel, Y. A. (2009) A similarity in the O-acetylation pattern of the O-antigens of Shigella flexneri types 1a, 1b and 2a, Carbohydr. Res., 344, 687-692.
22.Wang, J., Knirel, Y. A., Lan, R., Senchenkova, S. N., Luo, X., Perepelov, A. V., Wang, Y., Shashkov, A. S., Xu, J., and Sun, Q. (2014) Identification of an O-acyltransferase gene (oacB) that mediates 3- and 4-O-acetylation of rhamnose III in Shigella flexneri O antigens, J. Bacteriol., 196, 1525-1531.
23.Knirel, Y. A., Luo, X., Wang, J., Senchenkova, S. N., Lan, R., Shpirt, A. M., Du, P., Shashkov, A. S., Xu, J., and Sun, Q. (2014) Genetic and structural identification of an O-acyltransferase gene (oacC) responsible for the 3/4-O-acetylation on rhamnose III in Shigella flexneri serotype 6, BMC Microbiol., 14, 266.
24.Sun, Q., Knirel, Y. A., Wang, J., Luo, X., Senchenkova, S. N., Lan, R., Shashkov, A. S., and Xu, J. (2014) Serotype-converting bacteriophage SfII encodes an acyltransferase protein that mediates 6-O-acetylation of GlcNAc in Shigella flexneri O-antigens, conferring on the host a novel O-antigen epitope, J. Bacteriol., 196, 3656-3666.
25.Jakhetia, R., Marri, A., Stahle, J., Widmalm, G., and Verma, N. K. (2014) Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage, Sf101, from a serotype 7a strain, BMC Genomics, 15, 742.
26.Perepelov, A. V., L'vov, V. L., Liu, B., Senchenkova, S. N., Shekht, M. E., Shashkov, A. S., Feng, L., Aparin, P. G., Wang, L., and Knirel, Y. A. (2009) A new ethanolamine phosphate-containing variant of the O-antigen of Shigella flexneri type 4a, Carbohydr. Res., 344, 1588-1591.
27.Sun, Q., Knirel, Y. A., Lan, R., Wang, J., Senchenkova, S. N., Jin, D., Shashkov, A. S., Xia, S., Perepelov, A. V., Chen, Q., Wang, Y., Wang, H., and Xu, J. (2012) A novel plasmid-encoded serotype conversion mechanism through addition of phosphoethanolamine to the O-antigen of Shigella flexneri, PLoS ONE, 7, e46095.
28.Knirel, Y. A., Lan, R., Senchenkova, S. N., Wang, J., Shashkov, A. S., Wang, Y., Perepelov, A. V., Xiong, Y., Xu, J., and Sun, Q. (2013) O-antigen structure of Shigella flexneri serotype Yv and effect of the lpt-O gene variation on phosphoethanolamine modification of S. flexneri O-antigens, Glycobiology, 23, 475-485.
29.Sun, Q., Lan, R., Wang, J., Xia, S., Wang, Y., Wang, Y., Jin, D., Yu, B., Knirel, Y. A., and Xu, J. (2013) Identification and characterization of a novel Shigella flexneri serotype Yv in China, PLoS ONE, 8, e70238.
30.Kenne, L., Lindberg, B., Petersson, K., and Romanowska, E. (1977) Basic structure of the oligosaccharide repeating-unit of the Shigella flexneri O-antigens, Carbohydr. Res., 56, 363-370.
31.Carlin, N. I. A., and Lindberg, A. A. (1987) Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 strains, Infect. Immun., 55, 1412-1420.
32.Carlin, N. I. A., Bundle, D. R., and Lindberg, A. A. (1987) Characterization of five Shigella flexneri variant Y-specific monoclonal antibodies using defined saccharides and glycoconjugate antigens, J. Immunol., 138, 4419-4427.
33.Wang, J., Lan, R., Knirel, Y. A., Luo, X., Senchenkova, S. N., Shashkov, A. S., Xu, J., and Sun, Q. (2014) Serological identification and prevalence of a novel O-antigen epitope linked to 3- and 4-O-acetylated rhamnose III of lipopolysaccharide in Shigella flexneri, J. Clin. Microbiol., 52, 2033-2038.
34.Shashkov, A. S., Senchenkova, S. N., Sun, Q., Lan, R., Wang, J., Perepelov, A. V., Knirel, Y. A., and Xu, J. (2013) Structure of the O-antigen of a novel Shigella flexneri serotype, 1d (I: 7,8), Carbohydr. Res., 373, 93-96.
35.Kenne, L., Lindberg, B., Petersson, K., Katzenellenbogen, E., and Romanowska, E. (1977) Structural studies of the Shigella flexneri variant X, type 5a and 5b O-antigens, Eur. J. Biochem., 76, 327-330.
36.Dmitriev, B. A., Knirel, Y. A., Sheremet, O. K., Shashkov, A. S., Kochetkov, N. K., and Hofman, I. L. (1979) Somatic antigens of Shigella. The structure of the specific polysaccharide of Shigella newcastle (Sh. flexneri type 6) lipopolysaccharide, Eur. J. Biochem., 98, 309-316.
37.Wehler, T., and Carlin, N. I. A. (1988) Structural and immunochemical studies of the lipopolysaccharide from a new provisional serotype of Shigella flexneri, Eur. J. Biochem., 176, 471-476.
38.Foster, R. A., Carlin, N. I. A., Majcher, M., Tabor, H., Ng, L.-K., and Widmalm, G. (2011) Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with Shigella flexneri provisional serotype Y394 (1c), Carbohydr. Res., 346, 872-876.
39.Kondakova, A. N., Vinogradov, E. V., Shekht, M. E., Markina, A. A., Lindner, B., L'vov, V. L., Aparin, P. G., and Knirel, Y. A. (2010) Structure of the oligosaccharide region (core) of the lipopolysaccharides of Shigella flexneri types 2a and 5b, Bioorg. Khim., 36, 396-399.
40.Kubler-Kielb, J., Vinogradov, E., Mocca, C., Pozsgay, V., Coxon, B., Robbins, J. B., and Schneerson, R. (2010) Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates, Carbohydr. Res., 345, 1600-1608.
41.Guan, S., Bastin, D. A., and Verma, N. K. (1999) Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX, Microbiology, 145, 1263-1273.
42.Carlin, N. I. A., Wehler, T., and Lindberg, A. A. (1986) Shigella flexneri O-antigen epitopes: chemical and immunochemical analyses reveal that epitopes of type III and group 6 antigens are identical, Infect. Immun., 53, 110-115.
43.Lugowski, C., Romanowska, E., Kenne, L., and Lindberg, B. (1983) Identification of a trisaccharide repeating-unit in the enterobacterial common-antigen, Carbohydr. Res., 118, 173-181.
44.Dell, A., Oates, J., Lugowski, C., Romanowska, E., Kenne, L., and Lindberg, B. (1984) The enterobacterial common-antigen, a cyclic polysaccharide, Carbohydr. Res., 133, 95-104.
45.Carlin, N. I., Rahman, M., Sack, D. A., Zaman, A., Kay, B., and Lindberg, A. A. (1989) Use of monoclonal antibodies to type Shigella flexneri in Bangladesh, J. Clin. Microbiol., 27, 1163-1166.
46.Talukder, K. A., Dutta, D. K., Safa, A., Ansaruzzaman, M., Hassan, F., Alam, K., Islam, K. M., Carlin, N. I., Nair, G. B., and Sack, D. A. (2001) Altering trends in the dominance of Shigella flexneri serotypes and emergence of serologically atypical S. flexneri strains in Dhaka, Bangladesh, J. Clin. Microbiol., 39, 3757-3759.
47.Rusden, A. D., Stephenson, D. P., and Verma, N. K. (2013) Topological investigation of glucosyltransferase V in Shigella flexneri using the substituted cysteine accessibility method, Biochemistry, 52, 2655-2661.
48.Markine-Goriaynoff, N., Gillet, L., Van Etten, J. L., Korres, H., Verma, N., and Vanderplasschen, A. (2004) Glycosyltransferases encoded by viruses, J. Gen. Virol., 85, 2741-2754.
49.Mavris, M., Manning, P. A., and Morona, R. (1997) Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri, Mol. Microbiol., 26, 939-950.
50.Allison, G. E., Angeles, D., Tran-Dinh, N., and Verma, N. K. (2002) Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri, J. Bacteriol., 184, 1974-1987.
51.Allison, G. E., Angeles, D. C., Huan, P. T., and Verma, N. K. (2003) Morphology of temperate bacteriophage SfV and characterization of the DNA packaging and capsid genes: the structural genes evolved from two different phage families, Virology, 308, 114-127.
52.Sun, Q., Lan, R., Wang, Y., Wang, J., Wang, Y., Li, P., Du, P., and Xu, J. (2013) Isolation and genomic characterization of SfI, a serotype-converting bacteriophage of Shigella flexneri, BMC Microbiol., 13, 39.
53.George, D. T., Stephenson, D. P., Tran, E., Morona, R., and Verma, N. K. (2013) Complete genome sequence of SfII, a serotype-converting bacteriophage of the highly prevalent Shigella flexneri serotype 2a, Genome Announc., 1, e00626-13.
54.Jakhetia, R., Talukder, K. A., and Verma, N. K. (2013) Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri, BMC Genomics, 14, 677.
55.Sun, Q., Lan, R., Wang, Y., Wang, J., Luo, X., Zhang, S., Li, P., Wang, Y., Ye, C., Jing, H., and Xu, J. (2011) Genesis of a novel Shigella flexneri serotype by sequential infection of serotype-converting bacteriophages SfX and SfI, BMC Microbiol., 11, 269.
56.Stagg, R. M., Tang, S. S., Carlin, N. I., Talukder, K. A., Cam, P. D., and Verma, N. K. (2009) A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c, J. Bacteriol., 191, 6612-6617.
57.Thanweer, F., Tahiliani, V., Korres, H., and Verma, N. K. (2008) Topology and identification of critical residues of the O-acetyltransferase of serotype-converting bacteriophage, SF6, of Shigella flexneri, Biochem. Biophys. Res. Commun., 375, 581-585.
58.Casjens, S., Winn-Stapley, D. A., Gilcrease, E. B., Morona, R., Kuhlewein, C., Chua, J. E., Manning, P. A., Inwood, W., and Clark, A. J. (2004) The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging, J. Mol. Biol., 339, 379-394.
59.Sun, Q., Lan, R., Wang, Y., Wang, J., Xia, S., Wang, Y., Zhang, J., Yu, D., Li, Z., Jing, H., and Xu, J. (2012) Identification of a divergent O-acetyltransferase gene oac1b from Shigella flexneri serotype 1b strains, Emerg. Microbes Infect., 1, e21.
60.Sun, Q., Knirel, Y. A., Lan, R., Wang, J., Senchenkova, S. N., Shashkov, A. S., Wang, Y., Wang, Y., Luo, X., and Xu, J. (2014) Dissemination and serotype modification potential of pSFxv_2, an O-antigen PEtN modification plasmid in Shigella flexneri, Glycobiology, 24, 305-313.
61.Zhang, N., Lan, R., Sun, Q., Wang, J., Wang, Y., Zhang, J., Yu, D., Hu, W., Hu, S., Dai, H., Du, P., Wang, H., and Xu, J. (2014) Genomic portrait of the evolution and epidemic spread of a recently emerged multidrug-resistant Shigella flexneri clone in China, J. Clin. Microbiol., 52, 1119-1126.
62.Ye, C., Lan, R., Xia, S., Zhang, J., Sun, Q., Zhang, S., Jing, H., Wang, L., Li, Z., Zhou, Z., Zhao, A., Cui, Z., Cao, J., Jin, D., Huang, L., Wang, Y., Luo, X., Bai, X., Wang, P., Xu, Q., and Xu, J. (2010) Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri, J. Clin. Microbiol., 48, 419-426.
63.Qiu, S., Wang, Z., Chen, C., Liu, N., Jia, L., Liu, W., Wang, L., Hao, R., Zhang, L., Wang, Y., and Song, H. (2011) Emergence of a novel Shigella flexneri serotype 4s strain that evolved from a serotype X variant in China, J. Clin. Microbiol., 49, 1148-1150.

redpajama_set_name: RedPajamaCommonCrawl


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The Dominique Strauss-Kahn Rorschach Test
Leave a Comment / July 1, 2011 July 1, 2011
There's a great deal of disappointment, even distress, in the air as news spreads that Dominique Strauss-Kahn might not be charged with rape (or attempted rape, or sexual assault). He's guilty, the victim's character is being attacked in order to protect him, and the Culture of Rape will emerge triumphant once again — or so I'm being told by various Emails, Tweets, etc.
On the other hand, the whole thing was a conspiracy to facilitate the pillaging of the Greek people and the replacement of DSK at the IMF by a robber baron more loyal to the Austerity Agenda. Or so I have been assured.
In addition, the entire episode illustrates the danger in recklessly charging people — especially famous and important people — with crimes, until they have first been proven guilty. Or so we are learning from Thoughtful pundits.
I actually think that some (or none) or even all of these stories could be true. What I object to is the assertion that each one is true, not because the facts demonstrate it (we haven't been given the facts) but because that's the way the world is.
Here's a general depiction of the world that is true: Rape is common and traumatic and often goes unpunished; victims are put through further trauma when they press charges; honest victims are often viewed as liars; victims lacking legal status and union membership and other advantages can be even more reluctant to press charges; our culture does not sufficiently condemn rape; our mercenary firms fighting our wars can apparently rape their employees with legal immunity.
Here's something that depiction of the world doesn't tell us: DSK raped his accuser.
Here's another general depiction of the world that is true: False accusations of rape are extremely common. Acquittals and exonerations through DNA suggest a large number of false convictions in cases lacking DNA evidence. Americans used to lynch large numbers of African American men on the basis of false rape accusations. Well-intentioned opponents of date-rape have preached that regret can transform sex into rape.
Here's something that depiction of the world doesn't tell us: DSK's accuser lied about being raped.
Here's another general depiction of the world that is true: Major financial interests have no shame or conscience and will engage in all variety of dirty tricks to get their way. General Motors tried to set Ralph Nader up with a phony rape charge. DSK's downfall was perfectly timed to benefit the plutocrats and destroy the hopes of the Greek people.
Here's something that does not tell us: DSK was set up by the global financial oligarchs.
Here's yet another state of affairs that is true: DSK has both a suspicious history of numerous allegations of sexual harassment and assault and a great deal of financial influence himself. If any dirt could be found to defame his accuser (since no means could be found to buy her off) and if the prosecutors didn't find it on their own, DSK was far more likely to find and provide it than your average person accused, rightly or wrongly, of a crime.
Here's something that doesn't tell us: DSK is getting away with rape due to the unfairness of the U.S. legal system.
Of course, we might very well know one or more of these things to be indisputably true by the end of the day or the week or the month. Or we might never know. But a great many people are convinced that they already know; and that's what worries me.
There are international conspiracies. But there isn't always one everywhere. There are rapes. But there isn't always one everywhere. There are false accusations. But some accusations are true.
I get the impression that for many people these facts are theoretically recognizable but practically irrelevant.
A Vision for 2050
Leave a Comment / May 21, 2005 May 21, 2005
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Multiple national-award nominations for Norfolk hospital radio station
Emily Prince
Published: 2:22 PM March 2, 2019
Hospital Broadcasting Association award nominees Carrie Ingram-Gettins and Liam Mandy from Hospital Radio Lynn. Photo: HRL - Credit: HRL
A hospital radio station is celebrating multiple nominations at this year's Hospital Broadcasting Association (HBA) awards.
Volunteers at Hospital Radio Lynn based at the Queen Elizabeth Hospital, present daily radio shows and their dedication has been rewarded with more nominations at the annual awards which celebrate the best and brightest that the country has to offer for local hospital radio.
Liam Mandy and Carrie Ingram-Gettins have been nominated for Best Newcomer and Best Female respectively and will represent the station and county at the award ceremony in Stoke-on-Trent on March 30.
Miss Ingram-Gettins said: 'It means a lot to be nominated as it shows that the effort we put in pays off and our enthusiasm for the station comes across.'
Mr Mandy said: 'I'm incredibly honoured to be nominated for best newcomer. I've been with Hospital Radio Lynn for just over six months now and I've already learnt so much and met some wonderful people.'
For more information visit www.hospitalradiolynn.org.uk or click here


Released on February 15, 1962: (running time 1 hour and 23 minutes) A mad doctor keeps his girlfriend's head alive after she was beheaded in a car crash, and searches for a new body for her.
Doctor Bill Cortner, played by Jason Evers, is a modern doctor in 1962 who is experimenting with life and death. He is quietly stealing body parts from the hospital that he works at, so that he can experiment with creating a new person from the discarded body parts of others. He has had some success, but at the cost of many failures.
When his beautiful girlfriend Jan is beheaded in a horrible car accident, the good doctor decides to keep her head alive with his latest serum. Her head does indeed come to life as a result of his medical magic, but now he has only a day or so to find a body to graft onto her head. With murder on his mind, he heads into the city in search of a body. He must find a beautiful woman who can disappear without casting any suspicion on him. His search begins at a burlesque bar, and while the beauties inside lust for his wallet, he only has one thing on his mind – Murder.
Meanwhile, the head of his girlfriend is evolving, thanks to the magical serum that is keeping her alive. She discovers that one of her boyfriend's horrible failures is locked in a closet across the room. As her brain is evolving, she discovers that she can communicate with the monster in the closet. Her brain is becoming more powerful than the mightiest man alive, and she is becoming passionate about one thing – Murder.
So, while her boyfriend is on the hunt for a beautiful woman with a body to die for, her head is living in a pan of chemicals and, like him, is thinking about murder, - his murder. Pop a big bowl of white kernel popcorn with plenty of warm melted butter drizzled over it and enjoy the show.


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Tag: Sex Crimes
Outrage After Judge Reverses Teen's Rape Conviction, Says 5 Months in Jail Is 'Plenty'
Earlier this month, an Illinois judge reversed an 18-year-old man's sexual assault conviction — finding that the 148 days he spent in jail…
Singer Lisa Gentile Accuses Chris Noth of Sexual Assault
A fourth woman has accused actor Chris Noth of sexual assault. Singer Lisa Gentile says she had known Noth, who was fired this…
Longtime CNN Producer Sexually Abused 9-Year-Old Girl, Then Bribed Her Family: Feds
A former CNN senior producer sexually abused a 9-year-old girl, then tried to bribe a family member for their silence, federal prosecutors alleged…
See It: Dad's Roommate Arrested After 6-year-old Girl Tests Positive for Sexually Transmitted Disease
An Arizona man is behind bars, accused of assaulting a young child who reportedly tested positive for a sexually transmitted disease (STD). According…
Christy Giles: Suspect in Beloved Model's Death Charged with Sexually Assaulting 4 Women
A California man arrested in connection with the deaths of model Christy Giles and architectural project manager Hilda Marcela Cabrales-Arzola, in now facing…
CNN Fires Longtime Producer Accused of Sexually Grooming & Abusing Young Girls
CNN announced on Tuesday that it has fired a senior producer days after he was arrested last week in Connecticut for grooming underage…


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The Entertainment Hunter™ – Call of Duty Modern Warfare
Matthew Hunter is a Seneca College student who lives with Asperger's Syndrome. He loves movies, video games and television, and has agreed to review his favourites (and not-so-favourites!) for AO York.
Here is Matthew's review of Call of Duty Modern Warfare.
Before we get started: I am doing a livestream on Twitch this Saturday November 2, at 11:00am. This is my first fundraiser as part of Extra Life, a foundation dedicated to help kids all over. All proceeds will go to Sick Kids, I've set a goal for $100, so please, come visit and donate to help sick children. Again, my Twitch channel is mdhunter99.
By Matthew Hunter
It all started with a couple of vague leaks, leaks led to the first trailer, and oh boy it was a good one. Set to Metallica's Enter Sandman the trailer shows off EVERYTHING the Modern Warfare series is based on, fast paced action, big loud guns, all the fun toys any military has at their disposal, and the thrill of the fight.
Now you could call me a Call Of Duty fan, I've played every game since Call Of Duty 3 13 years ago. Recently the franchise has kind of lost its identity. Moving from the modern times to a more futuristic path, although I mostly kept with it. To the fans who worry about this one, don't. COD is back and it's not pulling any punches.
This game had promised so much: intense action, a strong story filled with twists and turns, map variety in multiplayer, a new way to play (at least in this franchise), haunting images you won't soon forget, and a little something called Crossplay. I am ecstatic to say this game very much delivered.
WARNING: Graphic content
In recent times the COD campaigns have followed a certain theme. In Ghosts it was brotherhood and betrayal, in Infinite Warfare it was the bond between commander and soldier, like a father-son relationship. In Modern Warfare, the theme Infinity award went with is War Crimes. War isn't pretty, it's a bloody, brutal conflict with no winners, only survivors. The Geneva Convention banned certain weapons and methods from warfare they deemed 'too brutal', in this game, you will learn of some of them.
Chlorine gas, booby traps, the military butchering civilians: it's all here. In the first mission you are sent in to retrieve some weaponized chlorine gas, from then the conflicts only get more brutal. I'm ashamed to say I may have accidentally killed more civilians than the terrorists did in the second mission.
In the same mission you are sent on a "house call"; the same terrorists have holed up in a house in London. This is where the campaign shines like a golden hawk; these house calls are some of the most stressful, slow, claustrophobic parts of the game, you have to clear an entire house, room to room, in the smallest of spaces, with no lights, only with night vision goggles and a rifle. Enemies may hide around corners, under beds, pretend to surrender only to charge you with a knife or shoot you. Remember, this is something that police, military, and special forces have to do regularly, this is how SEAL Team 6 killed Osama Bin Laden, and these missions are numerous.
I do warn those who do play the campaign, it gets more brutal than this, you will be forced to make some difficult decisions, and be forced to watch difficult scenarios. In one mission you play as one of the protagonists as a kid, in their village as the Russian army, led by the main antagonist General Barkov, murder everyone present. This can be a crushing moment and you, an 8 year old middle eastern girl, are forced to murder a Russian soldier, while said soldier antagonizes and hunts you down.
I believe that Infinity Ward and the writing team made the right call in bringing attention to the horrors of war crimes; they do happen, and they are a brutal realization of military life. The campaign was solid from beginning to end, bringing some fan favourite characters back was a surprising but fun twist, it paced itself well and the number of call backs to previous games gave something extra to the long time fans of the franchise.
The multiplayer mode felt great, like a nice walk on a beach or hot chocolate after a day of sledding. A fun return to boots on the ground combat after a few years with Jetpacks and ninja movements, there are numerous new features, some of the best class customization I've seen in a good long time, wide open maps sprawling through city streets, train depots, deserts, large caves, and a new 'night' mode where everything is dark only lit up by few lights and night vision goggles.
A new game mode called Ground War brings a whole new way to play for the franchise, vehicles such as tanks and helicopters are available to use. People say this is ripping off Battlefield, and yes it is, but it feels better.
There's not much else to say about the multiplayer mode. It's fun, but not revolutionary. What is revolutionary however is the implementation of Crossplay. Crossplay is the idea that the four main platforms of gaming can play together, Xbox, PlayStation, Nintendo, and PC. This is an experiment, but it's working so far.
Spec Ops:
This mode has been around for a decade now, and if I'm being honest, it isn't too notable in this game. But one thing that really stops this game from being fantastic is the lack of the Survival game mode from PC and Xbox. For an entire year the mode will only be available on PS4, until October 20 2020, likely when a new Call Of Duty will be released.
I love this game, it's a lot of fun, it can be difficult, exhilarating, and forgiving all the bugs, I'd say it's the best Call Of Duty in years.
Follow Matthew on Twitter at @mdhunter99
Matthew's on Twitch at mdhunter99 (language warning)
2019-11-01T22:51:33-04:00By Laura Webb|
Transitioning Together
The Entertainment Hunter™ – Call of Duty – Modern Warefare 2
#ThisIsWhyWeHoedown!
The Entertainment Hunter™ – Toy Story 4
The Entertainment Hunter™ – The Longest Day
My Truth – Marcus C.


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Is anyone else like this occasionally? I am right now. We played two huge 4 player games yesterday, and two the day before.
The worst I've been was after an all-day tourney we went to over the summer. Once I got home, I didn't play Heroscape for a week, and I didn't even go on Heroscapers for a few days!
I dunno. Maybe I'm just a loser, but I've got to take a break.


The Christchurch City Council has decided the city's water will be chlorinated for the next year.
Earlier this week the Christchurch City revealed engineers concerns that some of the below ground wellheads could become contaminated, especially during heavy flooding.
Council staff have recommended that temporary chlorination be installed at all of the city's 56 pump stations, at an initial cost of $600,000.
The damaged wellheads will also be repaired.
Today the council voted 12-3 in favour of chlorinating the water supply.
Christchurch Mayor Lianne Dalziel said even though the risk of contamination was low, there would be huge consequences if there was a problem.
Ms Dalziel said if there was a water crisis like the gastro outbreak in Havelock north in 2016, which landed dozens in hospital, hundreds of thousands of Christchurch residents could be infected.
Most councillors reluctantly supported the move.
"It's the last thing I ever thought we would agree too," said councillor Pauline Cotter.
"I would love not to, but as someone who has a young daughter, we need to do the responsible thing," councillor Yani Johanson said.
"It's our responsibility to provide safe drinking water, especially to the [city's] vulnerable," councillor Anne Galloway said.
However, some councillors opposed the move to chlorinate.
"I totally oppose adding chemicals to our pristine water," said councillor Mike Davidson, who received a round of applause from some members of the public.
"Our water is perfect, it has been through a lot, but it is still perfect."
Councillor Sara Templeton wanted to delay a decision until the council could consult the public on the issue.
Councillor Aaron Keown also opposed chlorination.
It will take 60 days to set up the chlorination systems.
After the meeting, Ms Dalziel said nobody wanted to see Christchurch's pristine water chlorinated.
"But when we are faced with a situation where we can no longer rely on the secure status of our groundwater because of the standard of the bore heads, and the fact that the engineers won't sign off on them, what else can we do as a council?"
Ilam MP Gerry Brownlee said Christchurch was well-known for having high-quality drinking water, which did not smell or taste of chlorine.
A decision to chlorinate it should not be made lightly, he said.
"You're talking about mass-dosing people with chlorine. It is a poison, that's why it's in the water to kill off all the bugs."
However, Infrastructure group manager Ashley Harper yesterday said the gastro outbreak in Havelock North in 2016, which left dozens in hospital, changed people's minds.
"We are recommending that we take steps to ensure that doesn't happen in our district," he said.
In December last year, a report into the Havelock North gastro outbreak warned hundreds of thousands of New Zealanders - one in five people - were at risk of getting sick and universal treatment of drinking water was needed.
Several South Island councils are considering permanently chlorinating their water to ensure it remains safe to drink.
A leading freshwater ecologist is criticising planned chlorination of Christchurch's water supply as a last resort, or ambulance-at-the-bottom-of-a-cliff approach.


The first TT event of the 2017 season (TTL_01) takes place this Tuesday 25th April on the Black Bull 10 mile TT course. We will be using the usual HQ at Black Bull (1st left on R154 from M3 exit to Trim). The nearest WC facilities are in Batterstown at the garage or the pub.
Sign on will close at 19:00 and the first rider will be off at 19:15. Marshal briefing is at 19.00 sharp.
For further course details and information see document below.
Good luck to everyone and above all, obey the marshals, look after yourself and other riders, and let's all stay safe and do it all again next week. We also ask riders to show the utmost respect to the venue we will be using, so please Leave No Trace – take home any litter with you and use dressing room and toilets where provided.
Next story TT season gets under way.