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KidsPeace believes that it is helpful to our readers to reprint some Healing Magazine articles
that are as relevant today as when they were published in our Magazine.
This article discusses how parents of special needs students should strive to interact well with their children's teachers and is written by author and Autisim advocate Ellen Notbohm. It was
published in the Spring/Summer 2007 issue.
The View from the Other
Side of the Desk: Are you a 'challenging parent?'
What special education
teachers want you to know
by Ellen Notbohm
As transitions go, my son's
transition to middle school had been smoother than any parent with an
Individualized Education Plan (IEP) in hand could hope for. It had been a very
good year with very good teachers. But, as the year wound down with alarming
speed, the scheduling of the annual IEP meeting just wasn't happening. Repeated
requests – at increasing decibel level – to resource teachers went unresolved
amid scheduling problems, administrative issues, illnesses and other
roadblocks. When we finally did meet, five days before the end of the school
year, I told the excellent resource teacher only half-jokingly, "You're almost
there. Only five more days and then you are done with me."
And this excellent teacher
stopped in his tracks and looked at me with surprise. "Oh no," he said. "No. I have had some challenging
parents this year, and you are not one of them."
At that, it was my turn to
stop in my tracks. What, I wanted very much to know, constitutes a
"challenging" parent? It was too
intriguing a thought to leave on the table so, a few months later, we came back
to it. His very thoughtfully painted portrait of a "challenging" parent led me
to ask other special educators, teachers of students aged toddler to high
school across several different school districts, the same question. And, while
each came from his/her own unique situation, the common threads in their
thoughts were striking. A number of these common threads formed the basis for
my book Ten Things Your Student with Autism Wishes You Knew. Here then is the
view from the other side of the desk, the voice of your special education
Be team-oriented
A combative attitude does
not enhance our ability to make progress with your child. Our relationship
should be an alliance, not an adversarial face off. We are all here because of
the child; he or she is our common interest, and it is important not to lose
sight of that. It is not about me or you, or whether we like each other.
Give me the courtesy of a
clean slate. You may have had bad experiences with previous teachers or
schools, but putting past conflicts or issues onto me, coming in with guns
blazing before you even have a chance to get to know me or my program is
counterproductive. "This is what has happened in the past, and I expect the
same from you" is looking for trouble where it is possible that none exists.
There is a difference
between being assertive and being aggressive – and there is a cost. Teachers
appreciate parents who are knowledgeable, effective advocates for their
children. Knowing your rights and knowing the facts of your situation, and requesting
services and accommodations firmly but respectfully are light years removed
from being a fist-pounder.
We are not here for the
money or the recognition. We are here because we love these kids. In an ideal
world, I want to share with the parent any inside perspective I have about 'the
system' and how it affects decisions made about their child. But, if I sense in
any way that the parent will use the information in a way that comes back on me
or threatens my job, it is only natural that I will not share.
Undermining me undermines
your child's learning.
Communicating to your child that everything that is going wrong is the
school's fault undermines your child's ability to trust me, to comply with
necessary classroom boundaries and, ultimately, to learn.
All children, even special
needs children, need to assume some level of responsibility for their behavior
and its consequences. We are sometimes faced with parents who say, "I cannot
believe my child would do such a thing. It must be somebody else's fault. If
you had been doing this, he wouldn't have been doing that." Sometimes that's
the case. However, when a parent insists it is always the case, I need to
gently suggest that a closer look be taken at what is actually going on.
Step back and listen as
open-mindedly as possible when faced with information that makes your blood
pressure rise. It's very common for children to exhibit a different set of
behaviors at school than they do at home.
Having to be both teacher
and case manager can put me in a very difficult position. Especially in early
childhood education, it often falls on the teacher/case manager to identify the
fact that my particular classroom or program isn't the best fit for your child.
Please know that, when I
tell you we need to transition your child to a different setting, it isn't
because I 'don't like him.' Hear me as objectively as possible when I tell you
that he is struggling too hard in the current placement and would benefit from
a different setting, that we need to modify the Individualized Family Service
Plan (IFSP) or IEP and find a better environment.
Don't assume I know
everything about your child. I may only have the prior year's academic
information, and perhaps no personal information at all. Tell me anything you
think is important for me to know about your "whole child." Be a resource for
us, a bridge between programs.
Share with us what has worked or not worked with your child in the past.
We cannot do everything for
your child. Your child is entitled by law to a free and appropriate education
in the least restrictive setting. That is not equivalent to the best possible
education. Think of it this way: You get the Chevy; you don't get the Cadillac.
You get safe, reliable transportation, but you don't get the CD player and the
leather seats. It's a distinction many parents don't understand that special
education is intended to provide for adequate growth, not maximum possible
Federal law mandates that we
make sure that kids who have a disability are making adequate progress, as
defined and measured yearly in their IEPs. The idea behind it is that, without
accommodation, they wouldn't make adequate progress in general education, and
therefore would not be getting a free and appropriate public education.
Let's say you have a fifth
grader who is reading at a 2nd grade level. It happens; teachers commonly look
at their classes and see a developmental range, so there are kids who end up in
4th or 5th grade reading several grade levels behind. So we set a goal, in a calendar
year, for the child to make a year's growth, which is what his peers would
make. But he is still behind; he
is not catching up. In order for him to catch up, he would have to outpace his
peers. Some kids do that, but it's very difficult and not realistic.
We have many commitments to
multiple content areas. If we were to spend half the day on reading alone –
sure, we could catch the kid up. But that's not appropriate because we give up
everything else. And so we always have that discussion every year in an IEP
meeting. We have a certain amount of time. How do we set goals? How much time
do we need to meet each goal? How much are we going to be able to accomplish
given math, science, social studies, all of these other content areas required
and from which kids benefit?
Your child is not my only
student. When I am meeting with you, when we are in a discussion and
problem-solving mode – in that moment, your student is the only one I am
concerned about. But back in my classroom, I have anywhere from a few to a few
dozen other students in my caseload, and I have the scheduling restrictions
that naturally come with that caseload. It simply is not possible for the needs
of one child to dictate my entire day. Asking that of me is painful for both of
Early intervention works
Here is an extension of a
universal truth: The earlier the
better – and the better the earlier the better. Catch things early, intervene
well and include your family, not just the school. No one was ever sorry they
intervened early, but legions of families regret "waiting to see if he outgrows
See the positive in your
Have an honest understanding
of what the range of your child's disability means, but also recognize his
strengths. Too often, the most difficult parents to work with are the ones who
cannot see the positive qualities of their kid. Their focus is stuck on what
the child can't do. Perhaps they do not want to have a child with a disability.
Perhaps they are stuck in the grieving process. But, for the teacher, it is
very hard to deal with.
Promote independence. Help
your child learn to do things for himself, rather than doing them for him. Many
teachers are parents themselves and understand the time stress families are
under. But, whether it's homework or personal organization, expedience in the
moment will impede his learning to be independent in the long run. If you pack
and unpack his backpack for him every day, how will he learn the importance of
being organized, knowing where things are when they are needed, how to find
items or information? The parents who are most effective are the ones who teach
as well as parent. The two are synonymous.
© 2006 Ellen Notbohm. This
article is based on the book Ten Things Your Student With Autism Wishes You
Knew, a 2006 iParenting Media Award winner.
Ellen Notbohm is author
of Ten Things Every Child with
Autism Wishes You Knew, a ForeWord 2005 Book of the Year Honorable Mention
winner, and co-author of the award-winning 1001 Great Ideas for Teaching and Raising Children with Autism Spectrum
Disorders. For article reprint permission, book excerpts, to learn more or
to contact Ellen, please visit www.ellennotbohm.com.


text:
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subset: commoncrawl


text:
Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a "base composition signature" (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.
1. A method of identifying an unknown bioagent by producing an ionized bioagent characterizing amplification product comprising:
aligning a gene sequence from a plurality of bioagent genomes to form an alignment;
selecting a gene region from said alignment comprising a region of variability defined by no more than 5% sequence identity among said plurality of bioagent genomes, which is flanked by a first and a second conserved region, each having 80% to 100% sequence identity among said plurality of bioagent genomes;
identifying said first and second conserved regions as primer binding sites;
designing a pair of primers comprising a first primer hybridizable to said first conserved region and second primer hybridizable to said second conserved region such that a plurality of amplification products of lengths of about 46 to 166 nucleobases can be produced upon amplification of said gene region with said pair of primers, wherein at least 8 of said plurality of amplification products have unique base compositions, wherein said base compositions that are present in a database identify the number, but not the nucleic acid gene sequence order, of A residues, C residues, T residues, G residues, U residues, analogues thereof and mass tag residues thereof in said amplification products;
amplifying a nucleic acid from an unknown bioagent with said pair of primers to produce a bioagent characterizing amplification product;
ionizing said amplification product;
determining the base composition of said ionized amplification product;
comparing said base composition of said ionized amplification product with said database containing said base compositions of said plurality of amplification products; and
identifying said unknown bioagent using said base composition of said ionized amplification product without sequencing said ionized amplification product.
2. The method of
3. The method of
4. The method of
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6. The method of
7. The method of
This application is a continuation of U.S. Ser. No. 10/156,608 filed May 24, 2002, now U.S. Pat. No. 7,108,974 which is a divisional of U.S. Ser. No. 09/798,007 filed Mar. 2, 2001, now abandoned each which is incorporated herein by reference in its entirety.
This invention was made with United States Government support under DARPA contract MDA972-00-C-0053. The United States Government has certain rights in the invention.
The present invention relates to methods for rapid detection and identification of bioagents from environmental, clinical or other samples. The methods provide for detection and characterization of a unique base composition signature (BCS) from any bioagent, including bacteria and viruses. The unique BCS is used to rapidly identify the bioagent.
Rapid and definitive microbial identification is desirable for a variety of industrial, medical, environmental, quality, and research reasons. Traditionally, the microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through direct examination and culture of specimens. Since the mid-1980s, researchers have repeatedly demonstrated the practical utility of molecular biology techniques, many of which form the basis of clinical diagnostic assays. Some of these techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). These procedures, in general, are time-consuming and tedious. Another option is the polymerase chain reaction (PCR) or other amplification procedure which amplifies a specific target DNA sequence based on the flanking primers used. Finally, detection and data analysis convert the hybridization event into an analytical result.
Other techniques for detection of bioagents include high-resolution mass spectrometry (MS), low-resolution MS, fluorescence, radioiodination, DNA chips and antibody techniques. None of these techniques is entirely satisfactory.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. However, high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents ("cluttered" background). Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem. 69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998; Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need.
One embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the molecular mass of the amplification product; and (d) comparing the molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent. In one aspect of this preferred embodiment, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of this preferred embodiment, the bioagent is a bacterium, virus, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d). Preferably, the one or more molecular mass is contained in a database of molecular masses. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
Another embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the base composition of the amplification product; and (d) comparing the base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent. In one aspect of this preferred embodiment, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of this preferred embodiment, the bioagent is a bacterium, virus, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base composition signatures of amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d). Preferably, the one or more base compositions is contained in a database of base compositions. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
The present invention also provides a method for detecting a single nucleotide polymorphism in an individual, comprising the steps of (a) isolating nucleic acid from the individual; (b) contacting the nucleic acid with oligonucleotide primers which hybridize to regions of the nucleic acid which flank a region comprising the potential polymorphism; (c) amplifying the region to produce an amplification product; (d) determining the molecular mass of the amplification product; and (e) comparing the molecular mass to the molecular mass of the region in an individual known to have the polymorphism, wherein if the molecular masses are the same then the individual has the polymorphism.
In one aspect of this preferred embodiment, the primers hybridize to highly conserved sequences. Preferably, the polymorphism is associated with a disease. Alternatively, the polymorphism is a blood group antigen. In one aspect of the preferred embodiment, the amplifying step is polymerase chain reaction. Alternatively, the amplification step is ligase chain reaction or strand displacement amplification. Preferably, the amplification product is ionized prior to mass determination. In one aspect, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.
The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with PCR-based BCS technology using "intelligent primers" which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The high-resolution MS technique is used to determine the molecular mass and base composition signature (BCS) of the amplified sequence region. This unique "base composition signature" (BCS) is then input to a maximum-likelihood detection algorithm for matching against a database of base composition signatures in the same amplified region. The present method combines PCR-based amplification technology (which provides specificity) and a molecular mass detection mode (which provides speed and does not require nucleic acid sequencing of the amplified target sequence) for bioagent detection and identification.
The present method allows extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure. Thus, the method is useful in a wide variety of fields, including, but not limited to, environmental testing (e.g., detection and discrimination of pathogenic vs. non-pathogenic bacteria in water or other samples), germ warfare (allowing immediate identification of the bioagent and appropriate treatment), pharmacogenetic analysis and medical diagnosis (including cancer diagnosis based on mutations and polymorphisms; drug resistance and susceptibility testing; screening for and/or diagnosis of genetic diseases and conditions; and diagnosis of infectious diseases and conditions). The method leverages ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives.
The present method can be used to detect and classify any biological agent, including bacteria, viruses, fungi and toxins. As one example, where the agent is a biological threat, the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes. In addition, the methods can be used to identify natural or deliberate engineering events including chromosome fragment swapping, molecular breeding (gene shuffling) and emerging infectious diseases.
Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species (Proc. Natl. Acad. Sci. U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins are DNA polymerase III beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G. Operons can also be targeted using the present method. One example of an operon is the bfp operon from enteropathogenic E. coli. Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential. The method can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
A theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor. The complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved. However, the present invention provides a straightforward strategy for obtaining information with the same practical value using base composition signatures (BCS). While the base composition of a gene fragment is not as information-rich as the sequence itself, there is no need to analyze the complete sequence of the gene if the short analyte sequence fragment is properly chosen. A database of reference sequences can be prepared in which each sequence is indexed to a unique base composition signature, so that the presence of the sequence can be inferred with accuracy from the presence of the signature. The advantage of base composition signatures is that they can be quantitatively measured in a massively parallel fashion using multiplex PCR (PCR in which two or more primer pairs amplify target sequences simultaneously) and mass spectrometry. These multiple primer amplified regions uniquely identify most threat and ubiquitous background bacteria and viruses. In addition, cluster-specific primer pairs distinguish important local clusters (e.g., anthracis group).
In the context of this invention, a "bioagent" is any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, toxin genes and bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.
As used herein, a "base composition signature" (BCS) is the exact base composition from selected fragments of nucleic acid sequences that uniquely identifies the target gene and source organism. BCS can be thought of as unique indexes of specific genes.
As used herein, "intelligent primers" are primers which bind to sequence regions which flank an intervening variable region. In a preferred embodiment, these sequence regions which flank the variable region are highly conserved among different species of bioagent. For example, the sequence regions may be highly conserved among all Bacillus species. By the term "highly conserved", it is meant that the sequence regions exhibit between about 80-100%, more preferably between about 90-100% and most preferably between about 95-100% identity. Examples of intelligent primers which amplify regions of the 16S and 23S rRNA are shown in
One main advantage of the detection methods of the present invention is that the primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same primer pair can be used to identify any desired bacterium because it will bind to the conserved regions which flank a variable region specific to a single species, or common to several bacterial species, allowing nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition. For example, the 16S—971-1062, 16S—1228-1310 and 16S—1100-1188 regions are 98-99% conserved in about 900 species of bacteria (16S=16S rRNA, numbers indicate nucleotide position). In one embodiment of the present invention, primers used in the present method bind to one or more of these regions or portions thereof.
The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with nucleic acid amplification-based BCS technology using "intelligent primers" which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s). Although the use of PCR is preferred, other nucleic acid amplification techniques may also be used, including ligase chain reaction (LCR) and strand displacement amplification (SDA). The high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments. The resolved spectral lines are then translated to BCS which are input to a maximum-likelihood detection algorithm matched against spectra for one or more known BCS. Preferably, the bioagent BCS spectrum is matched against one or more databases of BCS from vast numbers of bioagents. Preferably, the matching is done using a maximum-likelihood detection algorithm.
In a preferred embodiment, base composition signatures are quantitatively measured in a massively parallel fashion using the polymerase chain reaction (PCR), preferably multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids must be present for detection of bioagents by MS. A wide variety of techniques for preparing large amounts of purified nucleic acids or fragments thereof are well known to those of skill in the art. PCR requires one or more pairs of oligonucleotide primers which bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer. The primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Taq polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis. The solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence.
The "intelligent primers" define the target sequence region to be amplified and analyzed. In one embodiment, the target sequence is a ribosomal RNA (rRNA) gene sequence. With the complete sequences of many of the smallest microbial genomes now available, it is possible to identify a set of genes that defines "minimal life" and identify composition signatures that uniquely identify each gene and organism. Genes that encode core life functions such as DNA replication, transcription, ribosome structure, translation, and transport are distributed broadly in the bacterial genome and are preferred regions for BCS analysis. Ribosomal RNA (rRNA) genes comprise regions that provide useful base composition signatures. Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes. For example, there are currently over 13,000 sequences in the ribosomal RNA database that has been created and maintained by Robin Gutell, University of Texas at Austin, and is publicly available on the Institute for Cellular and Molecular Biology web page (rna(dot)icmb(dot)utexas(dot)edu/). There is also a publicly available rRNA database created and maintained by the University of Antwerp, Belgium at rrna(dot)uia(dot)ac(dot)be.
These databases have been analyzed to determine regions that are useful as base composition signatures. The characteristics of such regions are: a) between about 80 and 100%, preferably >about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, preferably no more than about 50-250 nucleotides, and more preferably no more than about 60-100 nucleotides, between the conserved regions.
Due to their overall conservation, the flanking rRNA primer sequences serve as good "universal" primer binding sites to amplify the region of interest for most, if not all, bacterial species. The intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature.
It is advantageous to design the "intelligent primers" to be as universal as possible to minimize the number of primers which need to be synthesized, and to allow detection of multiple species using a single pair of primers. These primer pairs can be used to amplify variable regions in these species. Because any variation (due to codon wobble in the 3rd position) in these conserved regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal base". For example, under this "wobble" pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) or the purine analog 1-(2-deoxy-□-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res. 24:3302-3306, 1996).
In another embodiment of the invention, to compensate for the somewhat weaker binding by the "wobble" base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs are 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G. Propynes are described in U.S. Pat. Nos. 5,645,985, 5,830.653 and 5,484,908, the entire contents of which are incorporated herein by reference. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, the entire contents of which are incorporated herein by reference. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, the entire contents of which are incorporated herein by reference.
Bacterial biological warfare agents capable of being detected by the present methods include Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders), Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, and Vibrio cholerae.
Besides 16S and 23S rRNA, other target regions suitable for use in the present invention for detection of bacteria include 5S rRNA and RNase P (
Biological warfare fungus biowarfare agents include coccidioides immitis (Coccidioidomycosis).
Biological warfare toxin genes capable of being detected by the methods of the present invention include botulism, T-2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin and saxitoxin.
Biological warfare viral threat agents are mostly RNA viruses (positive-strand and negative-strand), with the exception of smallpox. Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high. RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases.
Examples of (−)-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbillivirus).
Examples of (+)-strand RNA viruses include picornaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross River virus, Venezuelan equine encephalitis virus, Rubella virus, hepatitis E virus). The hepatitis C virus has a 5′-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3′-untranslated region of 240 nucleotides. The 5′-UTR and 3′-UTR are 99% conserved in hepatitis C viruses.
In one embodiment, the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a (−)-strand RNA virus. (+)-strand RNA viruses are double stranded RNA and replicate by RNA-directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template. Helicase unwinds the RNA duplex to allow replication of the single stranded RNA. These viruses include viruses from the family picornaviridae (e.g., poliovirus, coxsackievirus, echovirus), togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae (e.g., lymphocytic choriomeningitis virus, lassa fever virus), cononaviridae (e.g., human respiratory virus) and Hepatitis A virus. The genes encoding these proteins comprise variable and highly conserved regions which flank the variable regions.
In a preferred embodiment, the detection scheme for the PCR products generated from the bioagent(s) incorporates three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a BCS that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel.
In one embodiment, the method can be used to detect the presence of antibiotic resistance and/or toxin genes in a bacterial species. For example, Bacillus anthracis comprising a tetracycline resistance plasmid and plasmids encoding one or both anthracis toxins (px01 and/or px02) can be detected by using antibiotic resistance primer sets and toxin gene primer sets. If the B. anthracis is positive for tetracycline resistance, then a different antibiotic, for example quinalone, is used.
Mass spectrometry (MS)-based detection of PCR products provides all of these features with additional advantages. MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product with a unique base composition is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons. Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). For example, MALDI of nucleic acids, along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.).
Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.
In general, the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS). In one embodiment of the invention, the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents). To achieve minimal latency, a continuous sample stream flows directly into the PGC-MS combustion chamber. When an increase in biomass is detected, a PCR process is automatically initiated. Bioagent presence produces elevated levels of large molecular fragments from 100-7,000 Da which are observed in the PGC-MS spectrum. The observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove (combining PCR and MS, preferably FT-ICR MS) is initiated. Optionally, alarms or other processes (halting ventilation flow, physical isolation) are also initiated by this detected biomass level.
The accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance 13C and 15N isotopes, and assignment of the charge state for any ion. The cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow. The latter two problems are addressed by operating with a resolving power of >100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA. The resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [M-14H+]14− charge state of an 84mer PCR product is poorly characterized and assignment of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components are visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward. The [13C,15N]-depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al., Nucl. Acids Res. 20:4515-4523, 1992).
While mass measurements of intact nucleic acid regions are believed to be adequate to determine most bioagents, tandem mass spectrometry (MSn) techniques may provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3′ terminus and a 5′ phosphate following internal cleavage) and the α-Base series (having an intact 5′ terminus and a 3′ furan).
The second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample.
If there are two or more targets of similar base composition or mass, or if a single amplification reaction results in a product which has the same mass as two or more bioagent reference standards, they can be distinguished by using mass-modifying "tags." In this embodiment of the invention, a nucleotide analog or "tag" is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses. Such tags are described in, for example, PCT WO97/33000. This further limits the number of possible base compositions consistent with any mass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined.
In another amplification reaction, the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C. The combination of the A/T reaction and G/C reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in
In another example, assume the measured molecular masses of each strand are 30,000.115 Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand.
Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing. The detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes.
Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels. The program emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database (e.g. GenBank) is used to define the mass basecount matched filters. The database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted the maximum likelihood process is applied to this "cleaned up" data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.
In one embodiment, a strategy to "triangulate" each organism by measuring signals from multiple core genes is used to reduce false negative and false positive signals, and enable reconstruction of the origin or hybrid or otherwise engineered bioagents. After identification of multiple core genes, alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and potential primer binding sites flanking variable regions are identified. Next, amplification target regions for signature analysis are selected which distinguishes organisms based on specific genomic differences (i.e., base composition). For example, detection of signatures for the three part toxin genes typical of B. anthracis (Bowen, J. E. and C. P. Quinn, J. Appl. Microbiol. 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
The present method can also be used to detect single nucleotide polymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly and accurately. A SNP is defined as a single base pair site in the genome that is different from one individual to another. The difference can be expressed either as a deletion, an insertion or a substitution, and is frequently linked to a disease state. Because they occur every 100-1000 base pairs, SNPs are the most frequently bound type of genetic marker in the human genome.
For example, sickle cell anemia results from an A-T transition, which encodes a valine rather than a glutamic acid residue. Oligonucleotide primers may be designed such that they bind to sequences which flank a SNP site, followed by nucleotide amplification and mass determination of the amplified product. Because the molecular masses of the resulting product from an individual who does not have sickle cell anemia is different from that of the product from an individual who has the disease, the method can be used to distinguish the two individuals. Thus, the method can be used to detect any known SNP in an individual and thus diagnose or determine increased susceptibility to a disease or condition.
In one embodiment, blood is drawn from an individual and peripheral blood mononuclear cells (PBMC) are isolated and simultaneously tested, preferably in a high-throughput screening method, for one or more SNPs using appropriate primers based on the known sequences which flank the SNP region. The National Center for Biotechnology Information maintains a publicly available database of SNPs (www.ncbi.nlm.nih.gov/SNP/).
The method of the present invention can also be used for blood typing. The gene encoding A, B or O blood type can differ by four single nucleotide polymorphisms. If the gene contains the sequence CGTGGTGACCCTT (SEQ ID NO:5), antigen A results. If the gene contains the sequence CGTCGTCACCGCTA (SEQ ID NO:6) antigen B results. If the gene contains the sequence CGTGGTACCCCTT (SEQ ID NO:7), blood group O results ("−" indicates a deletion). These sequences can be distinguished by designing a single primer pair which flanks these regions, followed by amplification and mass determination.
While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.
In one embodiment, nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry. Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al.). The nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
FTICR Instrumentation: The FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber. The spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute. The Bruker data-acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments. For oligonucleotides in the 20-30-mer regime typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z.
Modified ESI Source: In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator. The ESI ion optics consist of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz. A lab-built electromechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the "open" position via a TTL pulse from the data station. When in the "closed" position, a stable electrospray plume is maintained between the ESI emitter and the face of the shutter. The back face of the shutter arm contains an elastomeric seal which can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position. When the shutter is triggered, a "time slice" of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir. The rapid response time of the ion shutter (<25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir.
Apparatus for Infrared Multiphoton Dissociation: A 25 watt CW CO2 laser operating at 10.6 μm has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications. An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet. Using standard IR-compatible mirrors and kinematic mirror mounts, the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
Table 1 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms. The primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in
Included in the short bacterial database cross-section in Table 1 are many well known pathogens/biowarfare agents (shown in bold/red typeface) such as Bacillus anthracis or Yersinia pestis as well as some of the bacterial organisms found commonly in the natural environment such as Streptomyces. Even closely related organisms can be distinguished from each other by the appropriate choice of primers. For instance, two low G+C organisms, Bacillus anthracis and Staph aureus, can be distinguished from each other by using the primer pair defined by 16S—1337 or 23S—855 (M of 4 Da).
A conserved Bacillus region from B. anthracis (A14G9C14T9) and B. cereus (A15G9C13T9) having a C to A base change was synthesized and subjected to ESI-TOF MS. The results are shown in
In other examples of the present invention, the pathogen Vibrio cholera can be distinguished from Vibrio parahemolyticus with M>600 Da using one of three 16S primer sets shown in Table 2 (16S—971, 16S—1228 or 16S—1294) as shown in Table 4. The two mycoplasma species in the list (M. genitalium and M. pneumoniae) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced resolving power for closely related organisms. In cases such as Bacillus anthracis and Bacillus cereus that are virtually indistinguishable from each other based solely on mass differences, compositional analysis or fragmentation patterns are used to resolve the differences. The single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background.
Table 5 shows the expected molecular weight and base composition of region 16S—1100-1188 in Mycobacterium avium and Streptomyces sp.
Table 6 shows base composition (single strand) results for 16S—1100-1188 primer amplification reactions for different species of bacteria. Species which are repeated in the table (e.g., Clostridium botulinum) are different strains which have different base compositions in the 16S—1100-1188 region.
The same organism having different base compositions are different strains. Groups of organisms which are highlighted and in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example, Bacillus anthracis can be distinguished from Bacillus cereus and Bacillus thuringiensis using the primer 16S—971-1062 (Table 6). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g. anthracis cluster) are distinguished at high resolution with focused sets of primer pairs. The known biowarfare agents in Table 6 are Bacillus anthracis, Yersinia pestis, Francisella tularensis and Rickettsia prowazekii.
The sequence of B. anthracis and B. cereus in region 16S—971 is shown below. Shown in bold is the single base difference between the two species which can be detected using the methods of the present invention. B. anthracis has an ambiguous base at position 20.
The mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in
An ESI-TOF MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1228 region of the B. anthracis 16S rRNA gene. The results (
An ESI-FTICR-MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1337 region of the B. anthracis 16S rRNA gene. The results (
ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5 μM) from the saspB gene of B. anthracis containing an internal mass standard at an ESI of 1.7 μL/min as a function of sample consumption. The results (
An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standard was obtained following addition of 5 mM TBA-TFA buffer to the solution. This buffer strips charge from the oligonucleotide and shifts the most abundant charge state from [M-8H+]8− to [M-3H+]3− (
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Three individuals were arrested after failing to stop at a stop sign on Sunday night, August 12, at the intersection of HWY 3 and HWY 93 in Rattan.
Deputies Russell Bailey, Shawn Culver and James Marzek were involved with a traffic stop after they pulled over a ford pickup which they witnessed running stop sign in the early morning hours. Officers get the vehicle pulled over at the First Baptist Church in Rattan, directly across from Rattan Public Schools.
Officers noticed that there were three individuals in the vehicle, two males and one female. When the driver, later identified as Jeffrey Fromme, reached up to get his license and insurance information he flipped the sun visor down and a orange pill bottle fell into his lap. Officers noticed the pill bottles label had been pealed off. Inside the bottle were four white bar like pills that officers believed to be Xanax.
Officers also found an Altoids tin containing a green leafy substance that field tested as marijuana, a white smoking device that also contained the leafy substance, a glass smoking device that contained a white residue that tested positive for methamphetamine, open beer containers and a drinking glass that contained alcohol which was sitting in the cup holder.
The female passenger was identified as Callie Davis. When officers asked her if she had been drinking she just started laughing.
Fromme admitted that he had been drinking and that he had taken a hit off the pipe and had taken some Xanax while they were at a party earlier that night. Officers did a SFST (field sobriety test) on Fromme, which he did not pass.
The other male occupant of the vehicle was identified as Ryan Yates. Yates also told officers that he had been drinking and was also drinking the beer that was laying in the passenger side floor board of the truck.
All three individuals were placed under arrest and transported to Pushmataha County Jail.
Davis was charged with possession of controlled substance with 1000 feet of school or park; possession of marijuana; possession of paraphernalia; transporting an open container and public drunk.
Fromme was charged with possession of controlled substance within 1000 feet of school or park; possession of controlled dangerous substance; unlawful possession of CDS without a prescription; driving a motor vehicle while under the influence of alcohol and drugs and failure to stop at a stop sign.
Yates was charged with public drunk and transporting an open container.


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Presently, Canadians have one of the highest life expectancies in the world. On average, a 65 year old man can expect to live another 17 years and a 65 year old woman can expect to live an additional 21 years. 13 of those years will be in good health but 4-8 will be in poor health. These videos make the case for the importance of physical activity in minimizing chronic disease and maximizing quality of life as we age.
Health Sciences student, Billy Tran, shares his experience at the CCAA's 2014 Research to Action Conference at Western University.
Reconnecting cities and an aging population
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CRESTVIEW — Former Crestview High School band members are sought to join the new Big Red Machine Alumni and Friends Association.
The non-profit group, organized by former Crestview High band members Graham Fountain, Al Aplin, Theresa Fleming Riley and Lane Holland, will hold its first public performance Oct. 18.
Fountain said Choctaw and Fort Walton Beach high schools have similar band alumni programs that are successful in raising funds for their respective bands.
Organizers took the plans for an alumni support group to current band director Jody Dunn, who embraced the idea.
"We're hoping to have as many alumni and directors and staff members, and anybody else who can claim they've been a part of the Big Red Machine as possible," Dunn said.
Crestview Mayor David Cadle, former band director, and late director Louis Lindsay will be honored for their service to the band and community during the event, Fountain said.
The association has also invited the daughter and grandson of Romulus H. Thompson, the band's 1938 founder, Fountain said.
"Although we have no idea yet how many alums will be in attendance, over 800 invitations have been sent out to former band members," Fountain said.
Fountain, a class of 1979 member who was a band captain and played first chair tuba, said alumni band organizers look forward to helping celebrate the Big Red Machine's 75th anniversary.
"This recognition has special significance because the first band in Crestview was also the first public school band program in the newly formed Okaloosa County," Fountain said.
The Big Red Machine Alumni and Friends Association "will strive to raise funds from private resources through the means of alumni band members and other citizens who wish to see the benefits the band program provides our young people continue," the group stated in a press release.
Crestview High School band or band staff alumni are invited to join the new Big Red Machine Alumni & Friends Association. Contact Al Aplin, firstname.lastname@example.com, 305-7122 or Graham Fountain, email@example.com, 830-0002.
Alumni Band schedule:
Oct. 17: 6:30-8 p.m., practice, Crestview High band room
Oct. 18: 4-5 p.m., practice, Crestview High band room
Oct. 18: 5-6:15 p.m., Alumni Band invitation-only reception; 6:30-10 p.m., Alumni band and Big Red Machine halftime performance
Contact News Bulletin Arts & Entertainment Editor Brian Hughes at 850-682-6524 or email@example.com. Follow him on Twitter @cnbBrian.


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(Release: May 1, 2012) - This past spring, HCC said farewell to several retirees, who have collectively given 325 years of service to the college. Recent retirees included:
Dennis Carroll
Custodian/Security Associate
38 Years of Service
Dr. Thomas Clemens
Professor, History and Political Science
34 Years of Service
Lieba J. Cohen
Director, Institutional Advancement
24 Years of Service
Ellen Crofford
Buyer/Inventory Control Associate
9 Years of Service
Dr. Vaughn Crowl
Professor, Psychology
41 Years of Service
Brenda Hassinger
Professor and Education Coordinator, Medical Imaging Programs
37 Years of Service
Mildred Keith
Financial Aid Virtual Advisor
9 Years of Service
Beverly Kruger
Academic Testing Center Assistant
23 Years of Service
P. Chris Marschner
Manager, Technical Innovation Center
21 Years of Service
Dixie Myers
Professor, English
37 Years of Service
Dr. Marlys Palmer
Professor, Physical Education
32 Years of Service
Spring Ward
Associate Professor, History and Political Science
20 Years of Service
Retirees were honored at a special reception on May 14 in the Kepler Center.
Photo Caption:
Front row, from left: Millie Keith, Brenda Hassinger, and Marlys Palmer.
Back row, from left: Chris Marschner, Vaughn Crowl, and Tom Clemens.
Not pictured: Dennis Carroll, Lieba J. Cohen, Ellen Crofford, Beverly Kruger, Dixie Myers, and Spring Ward.


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Consumer Reports evaluated common cancer screenings and found there are some tests men don't really need.
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Mrs. Ethel Mae Hobson, 83, of Snider Road passed away Friday, November 2, 2012 at Yadkin Valley Community Hospital. She was born February 13, 1929 in Yadkin County to the late Thad and Lucy Sizemore Hobson. Mrs. Hobson was a beloved wife, mother, and homemaker for her family.
She worked several years at Fall Creek Elementary School and was an active member of Union Grove Baptist Church as long as her health permitted. She served the church in various ways including, Sunday School Teacher, Choir, Bible School, and the W.M.U.. In addition to her parents, she was also preceded in death by her loving husband, Clay Monroe Hobson, and a sister, Grace Marie Jester.
Surviving are her children, Dianne H. Brendle and husband, David, of Elkin; Felix Monroe Hobson, and wife, Patricia, of Yadkinville; Kelly Vance Hobson, and wife, Derinda, of Aberdeen, NC; grandchildren, Natalie Roxanne Hobson, and husband, Graham Kirkham of San Diego, Ca., Nathan Elliott Hobson, San Francisco, Ca., Julian Prescott Hobson, Los Angeles, Ca., Ledena H. Dye and husband, Scott, of Aberdeen, Collin Jay Hobson, of Aberdeen; sisters, Lois (Herbert) Brendle, Sherlie (David) Reece; brothers, Sonnie (Shirley) Hobson, Ralph (Jackie) Hobson.
Her funeral service will be held 2:00 PM Monday in the Gentry Family Chapel by Rev. Jack Gentry and Chaplain Hal Finney. Burial will follow in the Union Grove Baptist Church Cemetery.
The family will receive friends from 6:30-8:30 PM Sunday at Gentry Family Funeral Service in Yadkinville. Flowers will be accepted or memorials may be made to Mountain Valley Hospice, PO Box 1267, Yadkinville, NC 27055. Online condolences may be made to www.gentryfuneralservice.com.


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Sat., Nov. 14*
at Texas Stars
Cedar Park Center
7 p.m.
Sun., Nov. 15*
at Houston Aeros
Toyota Center
4 p.m.
*Comcast Network 100
All GAMES WILL BE TELECAST LIVE ON MY50 CHICAGO OR COMCAST CABLE
Log on to chicagowolves.com for broadcast schedule, or to watch or listen to the action live online
The Wolves have potted 22 goals in their last six outings after scoring 12 markers in their previous six matchups; Chicago ranks 14th in the AHL with an average of 2.77 goals per game.
--- Tim Stapleton: Potted his first multiple-goal game of the season, including the game-winning tally, on Nov. 4 at Milwaukee ... Paces the Wolves and shares fifth in the AHL with seven goals in 13 games this season ... Shares the team lead with 10 points ... Ranked first on the Toronto Marlies and tied for seventh in the AHL with 79 points in 70 outings last season ... Paced the Marlies with 28 goals and 51 assists ... Appeared in four NHL outings with the Toronto Maple Leafs, picking up his first career NHL goal on Feb. 28 at Ottawa
--- Chris Chelios: Dished out two assists on Nov. 4 at Milwaukee, his third mutliple-point outing of the season ... Paces Wolves defensemen and ranks fifth on the team with seven points (G, 6A) in seven games played since signing with Chicago on Oct. 20 ... Ranks second on the team with a +6 plus/minus rating ... Appeared in 28 NHL games with the Detroit Red Wings last season, accruing 18 penalty minutes ... Also tallied an assist and two penalty minutes in two AHL games with the Grand Rapids Griffins last year
--- Arturs Kulda: Tallied two points (G, A) in two games last week, including his third career multiple point game on Nov. 4 ... Has set a career-high with two goals and has registered four points in 11 contests this season ... Paces the Wolves and shares second in the AHL with a +10 plus/minus rating ...Tallied 15 points (G, 14A) and 59 penalty minutes in 57 contests with the Wolves during his first full professional season last year
--- Johnny Pohl: Collected three points (G, 2A) in two games this week, which includes his second goal of the season ... Shares seventh on the team with five points (2G, 3A) in seven games this season ... Recorded three goals and 25 points in 22 outings with Lugano of the Swiss League and five goals and 12 points in 12 matchups with Vastra Frolunda of the Swedish Elite League last season
--- The Wolves saw a franchise record penalty killing streak come to a halt on Nov. 4, when they allowed their first power-play goal in seven games; Chicago blanked opponents on the man advantage in six consecutive games (26 of 26) for the first time since Oct. 18 through Nov. 2, 2000; the team has thwarted 30 of its opponent's last 31 power-play attempts and ranks eighth in the league with an 88.0 efficiency rating on the penalty kill (44 of 50).
--- The Wolves are in the midst of a season-high six game road trip; Chicago, which notched its first road win of the season on Nov. 4 at Milwaukee, will play its first three-games-in-three-days set this weekend when the team travels through Texas with stops in San Antonio, Austin and Houston.
--- Right wing Spencer Machacek has been held scoreless in a season-high four games; the 21-year-old forward shares the team lead with 10 points and ranks second with seven assists.
--- Chicago outshot Grand Rapids 32-23 on Friday, the fourth straight contest in which the team has outgunned its foes.
--- The Wolves have just allowed two third period goals in their last eight games; Chicago is outscoring opponents 11-5 in the final frame this season.
--- Defenseman Scott Lehman has dished out an assist in each of his last two games after being held scoreless in his first seven outings this season.
--- Wolves forward Andrew Kozek will visit the Elmwood Park Library, 1 Conti Parkway, on Wed., Nov. 11, at 4 p.m., to kick off the team's "Read to Succeed" program, presented by Vista Health System and Pace. Members of the squad will make more than 30 library visits throughout the Chicago area this season for the program, which has reached more than 500,000 children since its inception in 1995.
Fri., Nov. 6
(at) Grand Rapids 5, Chicago 4
--- The Wolves jumped out to a 2-0 lead, 6:50 into the game, but watched it slip away when they surrendered three goals in the second period on the way to a 5-4 loss to the Grand Rapids Griffins at Van Andel Arena
--- Forwards Brett Sterling, Andrew Kozek, Johnny Pohl and Joey Crabb each potted tallies for Chicago
--- Goaltender Manny Legace posted 10 saves on 14 shots in 28:07 of action, while Drew MacIntyre stopped eight of nine shots he faced in 30:30, taking the eventual loss.
Wed., Nov. 4
Chicago 4, (at) Milwaukee 3
--- Right wing Tim Stapleton broke a 3-3 tie midway through the third period to give the Wolves their first road win of the season, 4-3, over the Milwaukee Admirals at the Bradley Center
--- STAPLETON tallied twice, while Anthony Stewart and Arturs Kulda added the other markers
--- Goaltender Drew MacIntyre posted 14 saves in his third straight win


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Thursday, November 7, 2013
For the first time this season, the end result of a Stratford football game was less than a victory on Friday night.
The Knights traveled to South Florence for a battle of unbeatens and came back with a 26-14 defeat on their resume, their lone blemish in 10 tries this fall. Rainy conditions seemed to affect South Florence less.
South Florence quarterback Nick Jones had a hand in all four scores the Bruins (10-0), who scored 26 unanswered points after Taylor Morrison's 60-yard fumble return for a score gave Stratford a 7-0 lead in the first quarter.
Jones, who had 260 yards passing, brought South Florence within 7-6 on a 6-yard run then tossed touchdown passes of 17, 63 and 35 yards as the Bruins avenged a 31-14 loss in 2012 to Stratford.
Tabari Hines made two of the three scoring grabs for South Florence.
Jameak Jones made the game closer with 5:25 left, hauling in a 27-yard touchdown pass from quarterback Jacob Park.
Park, a Georgia commitment, had a rare off night, finishing 11 of 35 for 132 yards.
The Knights carry a 9-1 record into Friday's epic home clash with rival Goose Creek.
The Gators are 10-0 after beating West Ashley 49-21.
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Views: 345
Dear Becca,
Your own personal well being is of the utmost importance, it might feel as if you're hiding who you are from your family by not discussing this with your parents, but you have an awful lot to lose if you were to be disowned.
I would suggest to find a community (such as ThinkAtheist) to discuss the troubles that you have, I'm sure that many people can give you support and comfort that your parents can't give you.
Personally, if I were you I would wait till you're less dependent on your parents for financial support to come out of whatever closet you're hiding in. It's a pity that you feel that you can't be who you are around your family, whether it is true or not that you might not be able to, the feeling that you can't does seem to me to imply that you fear that your mom is basically a bigger supporter of baby Jeebus then her own baby (you)?
I wish you the best, both in family and in love. You're young, you are finding out what it means to be "Becca", you can hide who you are from your family, but I would recommend to find people to talk about your feelings so that you don't have to hide to yourself who you are. Expressing how you feel and think are vital to your development, I do hope that ThinkAtheist can help you by providing you with a sense of community.
I'm considering opening up to my close friends, only one knows so far and even though she disagrees she supports me and that means a lot.
Becca, you seem like you've got this figured out fairly well. I'd like to offer one additional bit of advice, which you may have thought of already, but in case you haven't, here it is. The people you consider to be your close friends may change, and that change could happen abruptly and for less than pleasant reasons.
Why do I mention this? When I was your age my good friend Tom, who was raised as a Pentecostal Protestant, began to have doubts about his religion. (And that is putting it mildly.) He shared these doubts with me and a handful of other friends from school, including his good friend and next-door neighbor Harold. Like you, Tom wasn't in a position to rebel openly, so he rebelled privately and in the company of those he trusted.
Skipping ahead, Tom and Harold both ended up liking the same girl: Cindy. The week before junior prom, Harold had a conversation with Tom's mother, during which he turned over the music CDs, comic books, and other items he was holding onto for Tom. Harold showed up at the prom with Cindy. Tom didn't show up at all. The rest of us rarely ever saw Tom outside of school again and when we did see him at school he didn't look very happy. Harold insisted he had done it for Tom's own good.
Be aware that telling someone about your atheism means you are irrevocably granting that person tremendous power over your life for many years to come. It might seem unthinkable that someone you know and trust would ever hurt you deliberately, but that is exactly the reason why you should think about it carefully. That goes especially in dealing with friends who disagree with your atheism and might one day decide to take similar steps for "your own good".
Good luck, Becca.
Has she ever discovered some friend of hers was not of the right religion? If not atheist, then some different sect, maybe very different, or perhaps agnostic?
If so what was her reaction? It will be worse when it's her daughter and it's atheism, not just her friend Katie being a Mormon. If the reaction is bad, she might let the blood relationship overrule her desire to disapprove violently. But I would not bet on it. Atheism is way beyond the pale for many religious people.
Even if she did decide not to disown/shun you, then you can expect attempts to evangelize you for the rest of your life.
Lesson for life: Religion causes segregation and discrimination.
I agree with your decision not to come out until you move out. I haven't officially come out to my parents, and I'm 32. I think they know, because of the opinions I have about related topics, but I don't see any point to sitting them down and saying "I'm an atheist".
Love your family, and when they talk about their silly beliefs, laugh at them silently, but love them all the same. It's a shame you can't expect them to laugh it off if you came out, but we all put up with a lot of crap when it comes to family.
I am going to tell you what I told my younger brother when he came out to me--
Don't tell your mom until you are able to live on your own.
There is always a chance that she will be accepting and kind, but it's also possible that your life will be made a living hell for the next few years. It's a shame that you have to do things this way, and that you can't be honest about who you are right now. Luckily, you have access to supportive communities of people elsewhere. I hope this will help you get through until a time when you are able to come out to your mom, and I hope she accepts and loves you for who you are.
Best wishes.
You have an entire website full of people who see eye to eye with you. From the statement your mother made, I am going to assume that she is rather right wing and conservative. When you are 16 you are sort of dependent on your parents for support, shelter, food, etc. Sometimes a parent will abuse this and make it seem like they "own" you. I understand that this can be extremely frustrating. I have been in a similar situation, but trust me it will get better. If you cannot come out to your family, find a friend that you can trust with anything and talk to them. Having an outlet can be very relieving. I understand that you object to your families beliefs, but why? Get a notebook, or an online blog, and investigate all the beliefs and think about why you object to them. This too can be quite liberating, especially when people comment and agree with you, or give you more information. Learning, although difficult, is liberating and gratifying. Knowledge is power.
Good Luck to you!
There's no reason why you should tell anyone at all even after you move out. It's not important
I wouldnt say anything about it. At 16 you are a young adult and now you will be forming your own world view and your going to change it a million times.
Dont completely close your mind from the modern Christian message either because it really has some great instruction that fits everyone.
Take the good things and discard the nonsense because Atheists can be better Christians than Christians.
My version of that is "Dont Be an Asshole"
All the Best.


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This toddler bed allows you to make the proper & first transition from the crib to a twin or full size bed. It uses the same mattress as the crib and the guard rails on either side add security. Perfect for your big boy or girl!
Dimensions: 54.5"L x 30.5"W x 26"H
Note: Mattress not included.
Gift Wrap not available.