Biological Chemistry

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Hello everyone! This is a blog about biological chemistry. I am a graduate student getting my PhD in organic chemistry with a specific focus on bio-organic chemistry. Click on the links for more information about this blog!

Yo Pro-tip for how to piss off other people in your department when you go to grad school: When using departmental equipment (such as the mass spec facility in general or, oh I don’t know, the ESI-MS in particular) make sure to contaminate or clog the instrument when you use it and then don’t tell anyone so that the next person who uses it can waste a whole bunch of their precious sample that it took them weeks to make. 

— 1 month ago with 6 notes
#science  #fuck you 
DNA Alkylation
Cellular DNA is constantly subject to damage by various intracellular and extracellular chemicals. Among these chemicals are alkylating agents, which transfer an alkyl group to a position in DNA. These chemicals can be cytotoxic and mutagenic, as alkylation of DNA, from simple methylation to the transfer of bulky alkyl groups, can cause polymerase error as well as blocking or stalling of polymerases. This can result from distortions in the helical geometry brought about by the alkylated DNA lesion, or by an inability of the polymerase to replicate past the lesion, even in the absence of significant helical distortion. The nucleobase guanine is especially vulnerable to alkylation by electrophilic alkylating agents due to its electron-rich character.
The simplest alkylating agent is methyl methanesulfonate (MMS) which delivers an electrophilic methyl group to nucleophilic sites on DNA (A above). This occurs primarily at the N7 of deoxyguanine, but also occurs at other nucleophilic sites in DNA such as the N3 of deoxyadenosine and, to a lesser extent, at other nitrogens and oxygens on DNA bases. Alkylation by MMS is believed to exhibit its cytotoxicity by stalling the replication fork. Despite its widespread use in research settings, MMS is not used therapeutically due to its high mutagenicity and nonspecific cytotoxicity.
The first alkylating agents used in a therapeutic capacity were the nitrogen mustards. These agents were derived from the notorious chemical weapon, mustard gas, which contains a sulfur instead of a nitrogen. They were initially developed as chemical weapons, but their therapeutic potential was recognized by researchers at Yale University during World War Two. The accepted mechanism for cytotoxicity derives from DNA alkylation and crosslink formation by these agents. The electrophilicity of these agents is increased drastically by a phenomenon known as neighboring group participation. The intramolecular nucleophilic attack by the Nitrogen leads to the formation of the aziridinium cation pictured above in figure B. This increases the electrophilicity of the C1 carbon, making it more prone to nucleophilic attack and greatly enhancing the rate of nucleophilic attack by N7 of guanine. Remember that intramolecular reactions happen far more quickly than intermolecular reactions, meaning that formation of the aziridinium cation is much faster than nucleophilic attack of guanine or water on the primary alkyl halide. Nucleophilic attack by the N7 of guanine results in alkylation of the nucleobase. Subsequent formation of another aziridinium cation and attack by another guanine, results in the formation of a DNA crosslink. This can occur with a guanine on the same strand or on the opposite strand. These bulky crosslinks are a severe block to polymerases, resulting in a failure to replicate or transcribe DNA, leading to cell death via apoptosis if the lesion is not repaired. Many nitrogen mustards, such as cyclophosphamide and chlorambucil are still in use today.
References:
http://pubs.acs.org/doi/abs/10.1021/jo300351g
http://www.hindawi.com/journals/jna/2010/543531/
http://www.nature.com/nrclinonc/journal/v6/n11/fig_tab/nrclinonc.2009.146_F1.html
http://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent

DNA Alkylation

Cellular DNA is constantly subject to damage by various intracellular and extracellular chemicals. Among these chemicals are alkylating agents, which transfer an alkyl group to a position in DNA. These chemicals can be cytotoxic and mutagenic, as alkylation of DNA, from simple methylation to the transfer of bulky alkyl groups, can cause polymerase error as well as blocking or stalling of polymerases. This can result from distortions in the helical geometry brought about by the alkylated DNA lesion, or by an inability of the polymerase to replicate past the lesion, even in the absence of significant helical distortion. The nucleobase guanine is especially vulnerable to alkylation by electrophilic alkylating agents due to its electron-rich character.

The simplest alkylating agent is methyl methanesulfonate (MMS) which delivers an electrophilic methyl group to nucleophilic sites on DNA (A above). This occurs primarily at the N7 of deoxyguanine, but also occurs at other nucleophilic sites in DNA such as the N3 of deoxyadenosine and, to a lesser extent, at other nitrogens and oxygens on DNA bases. Alkylation by MMS is believed to exhibit its cytotoxicity by stalling the replication fork. Despite its widespread use in research settings, MMS is not used therapeutically due to its high mutagenicity and nonspecific cytotoxicity.

The first alkylating agents used in a therapeutic capacity were the nitrogen mustards. These agents were derived from the notorious chemical weapon, mustard gas, which contains a sulfur instead of a nitrogen. They were initially developed as chemical weapons, but their therapeutic potential was recognized by researchers at Yale University during World War Two. The accepted mechanism for cytotoxicity derives from DNA alkylation and crosslink formation by these agents. The electrophilicity of these agents is increased drastically by a phenomenon known as neighboring group participation. The intramolecular nucleophilic attack by the Nitrogen leads to the formation of the aziridinium cation pictured above in figure B. This increases the electrophilicity of the C1 carbon, making it more prone to nucleophilic attack and greatly enhancing the rate of nucleophilic attack by N7 of guanine. Remember that intramolecular reactions happen far more quickly than intermolecular reactions, meaning that formation of the aziridinium cation is much faster than nucleophilic attack of guanine or water on the primary alkyl halide. Nucleophilic attack by the N7 of guanine results in alkylation of the nucleobase. Subsequent formation of another aziridinium cation and attack by another guanine, results in the formation of a DNA crosslink. This can occur with a guanine on the same strand or on the opposite strand. These bulky crosslinks are a severe block to polymerases, resulting in a failure to replicate or transcribe DNA, leading to cell death via apoptosis if the lesion is not repaired. Many nitrogen mustards, such as cyclophosphamide and chlorambucil are still in use today.

References:

http://pubs.acs.org/doi/abs/10.1021/jo300351g

http://www.hindawi.com/journals/jna/2010/543531/

http://www.nature.com/nrclinonc/journal/v6/n11/fig_tab/nrclinonc.2009.146_F1.html

http://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent

— 1 month ago with 33 notes
#biology  #chemistry  #DNA  #DNA damage  #molecular biology  #science 
biologicalchemistry:

DNA Damage: 8-oxo-guanine
In its most general sense, the central dogma of molecular biology states that genetic information is passed from DNA to RNA to proteins. It therefore holds that as the carrier of genetic information in cells, the integrity of DNA is highly important for normal cell survival and function. Changes in DNA sequence can lead to the production of aberrant proteins, causing cell death or altered cell function. Sometimes these changes in function can be serious enough to cause tumorogenesis. Despite its stability to damaging processes such as hydrolytic cleavage, DNA is not immune to oxidative damage, much of which results from reaction with reactive oxygen species. Reactive oxygen species are generated as a result of normal cellular metabolism. Although antioxidants and specific enzymes dedicated to the purpose are able to scavenge many of these ROS, some of them persist long enough to react with DNA. As Guanine has the lowest oxidation potential of all of the DNA bases (Steenken 1997), it is the most susceptible to oxidative damage. Oxidation at the C8 position can give the product 8-oxo-guanine. 

Instead of hydrogen bonding with cytosine as guanine normally does, it can form a Hoogsteen base pair with adenine (shown above). Therefore, during DNA replication, DNA polymerase may mistakenly insert an adenosine opposite an 8-oxo-dG, resulting in a stable change in DNA sequence, a process known as mutagenesis.
Edit: My previous phrasing “…resulting in a change in DNA sequence that can potentially be mutagenic” was mistaken. It was meant to imply that this change in DNA sequence, depending on its location, may or may not have an effect on amino acid sequence or gene expression. Therefore, the cell may or may not be affected. However, to clarify, a change in DNA sequence is, by definition, mutagenic.
Stay tuned for future posts on oxidative DNA damage!
References:
Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-618
Greenberg, M. M.; Chem Res Toxicol, 1998. 11, 1235-1248


Reblogging because I made a minor edit

biologicalchemistry:

DNA Damage: 8-oxo-guanine

In its most general sense, the central dogma of molecular biology states that genetic information is passed from DNA to RNA to proteins. It therefore holds that as the carrier of genetic information in cells, the integrity of DNA is highly important for normal cell survival and function. Changes in DNA sequence can lead to the production of aberrant proteins, causing cell death or altered cell function. Sometimes these changes in function can be serious enough to cause tumorogenesis. Despite its stability to damaging processes such as hydrolytic cleavage, DNA is not immune to oxidative damage, much of which results from reaction with reactive oxygen species. Reactive oxygen species are generated as a result of normal cellular metabolism. Although antioxidants and specific enzymes dedicated to the purpose are able to scavenge many of these ROS, some of them persist long enough to react with DNA. As Guanine has the lowest oxidation potential of all of the DNA bases (Steenken 1997), it is the most susceptible to oxidative damage. Oxidation at the C8 position can give the product 8-oxo-guanine. 

image

Instead of hydrogen bonding with cytosine as guanine normally does, it can form a Hoogsteen base pair with adenine (shown above). Therefore, during DNA replication, DNA polymerase may mistakenly insert an adenosine opposite an 8-oxo-dG, resulting in a stable change in DNA sequence, a process known as mutagenesis.

Edit: My previous phrasing “…resulting in a change in DNA sequence that can potentially be mutagenic” was mistaken. It was meant to imply that this change in DNA sequence, depending on its location, may or may not have an effect on amino acid sequence or gene expression. Therefore, the cell may or may not be affected. However, to clarify, a change in DNA sequence is, by definition, mutagenic.

Stay tuned for future posts on oxidative DNA damage!

References:

Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-618

Greenberg, M. M.; Chem Res Toxicol, 1998. 11, 1235-1248

Reblogging because I made a minor edit

— 2 months ago with 46 notes
#science  #chemistry  #DNA  #biology  #molecular biology 
DNA Damage: 8-oxo-guanine
In its most general sense, the central dogma of molecular biology states that genetic information is passed from DNA to RNA to proteins. It therefore holds that as the carrier of genetic information in cells, the integrity of DNA is highly important for normal cell survival and function. Changes in DNA sequence can lead to the production of aberrant proteins, causing cell death or altered cell function. Sometimes these changes in function can be serious enough to cause tumorogenesis. Despite its stability to damaging processes such as hydrolytic cleavage, DNA is not immune to oxidative damage, much of which results from reaction with reactive oxygen species. Reactive oxygen species are generated as a result of normal cellular metabolism. Although antioxidants and specific enzymes dedicated to the purpose are able to scavenge many of these ROS, some of them persist long enough to react with DNA. As Guanine has the lowest oxidation potential of all of the DNA bases (Steenken 1997), it is the most susceptible to oxidative damage. Oxidation at the C8 position can give the product 8-oxo-guanine. 

Instead of hydrogen bonding with cytosine as guanine normally does, it can form a Hoogsteen base pair with adenine (shown above). Therefore, during DNA replication, DNA polymerase may mistakenly insert an adenosine opposite an 8-oxo-dG, resulting in a stable change in DNA sequence, a process known as mutagenesis.
Edit: My previous phrasing “…resulting in a change in DNA sequence that can potentially be mutagenic” was mistaken. It was meant to imply that this change in DNA sequence, depending on its location, may or may not have an effect on amino acid sequence or gene expression. Therefore, the cell may or may not be affected. However, to clarify, a change in DNA sequence is, by definition, mutagenic.
Stay tuned for future posts on oxidative DNA damage!
References:
Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-618
Greenberg, M. M.; Chem Res Toxicol, 1998. 11, 1235-1248

DNA Damage: 8-oxo-guanine

In its most general sense, the central dogma of molecular biology states that genetic information is passed from DNA to RNA to proteins. It therefore holds that as the carrier of genetic information in cells, the integrity of DNA is highly important for normal cell survival and function. Changes in DNA sequence can lead to the production of aberrant proteins, causing cell death or altered cell function. Sometimes these changes in function can be serious enough to cause tumorogenesis. Despite its stability to damaging processes such as hydrolytic cleavage, DNA is not immune to oxidative damage, much of which results from reaction with reactive oxygen species. Reactive oxygen species are generated as a result of normal cellular metabolism. Although antioxidants and specific enzymes dedicated to the purpose are able to scavenge many of these ROS, some of them persist long enough to react with DNA. As Guanine has the lowest oxidation potential of all of the DNA bases (Steenken 1997), it is the most susceptible to oxidative damage. Oxidation at the C8 position can give the product 8-oxo-guanine. 

image

Instead of hydrogen bonding with cytosine as guanine normally does, it can form a Hoogsteen base pair with adenine (shown above). Therefore, during DNA replication, DNA polymerase may mistakenly insert an adenosine opposite an 8-oxo-dG, resulting in a stable change in DNA sequence, a process known as mutagenesis.

Edit: My previous phrasing “…resulting in a change in DNA sequence that can potentially be mutagenic” was mistaken. It was meant to imply that this change in DNA sequence, depending on its location, may or may not have an effect on amino acid sequence or gene expression. Therefore, the cell may or may not be affected. However, to clarify, a change in DNA sequence is, by definition, mutagenic.

Stay tuned for future posts on oxidative DNA damage!

References:

Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-618

Greenberg, M. M.; Chem Res Toxicol, 1998. 11, 1235-1248

— 2 months ago with 46 notes
#biology  #chemistry  #science  #DNA  #molecular biology 
s-c-i-guy:

Antibiotics May Have Been Wrongly Prescribed for Influenza, CDC Finds
During the 2013 flu season, more people needed antiviral medications than got them, and antibiotics were inappropriately given to a large proportion of patients with influenza (a viral disease that is not helped by taking antibiotics), according to a new study.
The researchers found that nearly 30 percent of the flu patients who were treated during the 2012-2013 influenza season may have been prescribed unnecessary antibiotics instead of antiviral therapy.
The new study is based on medical information from nearly 6,800 patients in five U.S. states. The researchers looked at whether the patients who were at high risk of developing serious complications from the flu received antiviral medications as recommended.
The results showed that antiviral and antibiotic medications were prescribed inappropriately. Less than 20 percent of patients with flu symptoms who could have had benefited from antiviral medication because they were at high risk of developing complications actually received the medication. Among patients who were confirmed to have had influenza through laboratory tests, 16 percent were prescribed antivirals.
In contrast, antibiotics were prescribed more frequently, with 30 percent of lab-confirmed influenza patients receiving one of the three antibiotics the researchers looked for, according to the study published today (July 17) in the journal Clinical Infectious Diseases.
The findings suggest that during 2012–2013 flu season, clinicians prescribed antiviral medications to a relatively small percentage of patients in the clinic, for whom the medications were recommended, and missed potential opportunities to prevent more serious complications in these patients, the researchers said.
The Centers for Disease Control and Prevention recommends using antiviral medication to treat flu patients who are at higher risk for developing serious complications, for example, all hospitalized patients who are suspected to have influenza, young children, people ages 65 years and older, pregnant women, and people with certain medical conditions, such as asthma and heart disease. Early treatment with antivirals reduces the patient’s risk of developing complications.
Seasonal influenza results in thousands of hospitalizations in the United States each year and can cause even more serious outcomes. In the 2012-2013 flu season, 64 children died from the flu between September 2012 and February 2013. The CDC recommends yearly vaccination as the best defense against seasonal influenza.
In the new study, Dr. Fiona Havers and a team from the CDC and several other institutions examined prescription records, looking for two influenza antiviral drugs (oseltamivir and zanamivir) and three common antibiotics (amoxicillin-clavulanate, amoxicillin and azithromycin).
Of the 6,766 patients in the study, 1,825 patients were tested and confirmed to have influenza, and also had available medical records stating whether they had received an antibiotics prescription. Thirty percent, or 540 of these patients, had received an antibiotic, the researchers found.
About 1,020 of the patients with flu symptoms were considered at high risk for influenza complications. Less than 200 of these patients (19 percent) received antivirals.
When the researchers focused on the patients who were confirmed to have influenza and were at high risk of complications, they found that only 28 percent of them received an antiviral medication prescription and 24 percent received an antibiotic.
Antibiotics cannot treat influenza, which is caused by a virus. While some of the antibiotics may have been appropriate because patients may have also had bacterial infections in addition to influenza, it is likely most were unnecessary, and potentially contributed to the growing problem of antibiotic resistance, the researchers said.
The findings highlight the need to educate clinicians about the appropriate use of antivirals and antibiotics in patients who have symptoms of influenza, the researchers said.
"Continuing education on appropriate antibiotic and antiviral use is essential to improve health care quality," the researchers wrote in their study.
source

It is certainly correct that antibiotics have no effect on influenza and should not be prescribed unless the physician suspects that the person has developed a secondary infection. However, there is increasing evidence that neuraminidase inhibitors such as oseltamivir (Tamiflu) and zanamivir (Relenza) have little benefit for those infected with influenza. Although the proposed mechanism of action and in vitro data appeared very promising (http://biologicalchemistry.tumblr.com/post/71792740352/the-role-of-glycoproteins-in-influenza-infection-and), the clinical data appears to suggest otherwise. A recent meta analysis by the Cochrane Collaboration found that neuraminidase inhibitors reduced the duration of symptoms by only 16.8 hours in adults and had no effect on the development of serious complications. Additionally, oseltamivir was found to increase the rate of nausea and vomiting by approximately 5% and was also found to increase the frequency of psychiatric events compared to placebo. Zanamivir was found to have lower toxicity, likely due to its lower bioavailability. Although it has been suggested that the studies reviewed by the Cochrane Collaboration did not include enough subjects with severe illness or those at high risk of developing complications, the review seems to suggest that the benefits of neuraminidase inhibitors do not significantly outweigh the risks for many patients.
Sources
http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD008965.pub4/abstract;jsessionid=D0952B85723A29FF11B3087DB5F71938.f03t02

s-c-i-guy:

Antibiotics May Have Been Wrongly Prescribed for Influenza, CDC Finds

During the 2013 flu season, more people needed antiviral medications than got them, and antibiotics were inappropriately given to a large proportion of patients with influenza (a viral disease that is not helped by taking antibiotics), according to a new study.

The researchers found that nearly 30 percent of the flu patients who were treated during the 2012-2013 influenza season may have been prescribed unnecessary antibiotics instead of antiviral therapy.

The new study is based on medical information from nearly 6,800 patients in five U.S. states. The researchers looked at whether the patients who were at high risk of developing serious complications from the flu received antiviral medications as recommended.

The results showed that antiviral and antibiotic medications were prescribed inappropriately. Less than 20 percent of patients with flu symptoms who could have had benefited from antiviral medication because they were at high risk of developing complications actually received the medication. Among patients who were confirmed to have had influenza through laboratory tests, 16 percent were prescribed antivirals.

In contrast, antibiotics were prescribed more frequently, with 30 percent of lab-confirmed influenza patients receiving one of the three antibiotics the researchers looked for, according to the study published today (July 17) in the journal Clinical Infectious Diseases.

The findings suggest that during 2012–2013 flu season, clinicians prescribed antiviral medications to a relatively small percentage of patients in the clinic, for whom the medications were recommended, and missed potential opportunities to prevent more serious complications in these patients, the researchers said.

The Centers for Disease Control and Prevention recommends using antiviral medication to treat flu patients who are at higher risk for developing serious complications, for example, all hospitalized patients who are suspected to have influenza, young children, people ages 65 years and older, pregnant women, and people with certain medical conditions, such as asthma and heart disease. Early treatment with antivirals reduces the patient’s risk of developing complications.

Seasonal influenza results in thousands of hospitalizations in the United States each year and can cause even more serious outcomes. In the 2012-2013 flu season, 64 children died from the flu between September 2012 and February 2013. The CDC recommends yearly vaccination as the best defense against seasonal influenza.

In the new study, Dr. Fiona Havers and a team from the CDC and several other institutions examined prescription records, looking for two influenza antiviral drugs (oseltamivir and zanamivir) and three common antibiotics (amoxicillin-clavulanate, amoxicillin and azithromycin).

Of the 6,766 patients in the study, 1,825 patients were tested and confirmed to have influenza, and also had available medical records stating whether they had received an antibiotics prescription. Thirty percent, or 540 of these patients, had received an antibiotic, the researchers found.

About 1,020 of the patients with flu symptoms were considered at high risk for influenza complications. Less than 200 of these patients (19 percent) received antivirals.

When the researchers focused on the patients who were confirmed to have influenza and were at high risk of complications, they found that only 28 percent of them received an antiviral medication prescription and 24 percent received an antibiotic.

Antibiotics cannot treat influenza, which is caused by a virus. While some of the antibiotics may have been appropriate because patients may have also had bacterial infections in addition to influenza, it is likely most were unnecessary, and potentially contributed to the growing problem of antibiotic resistance, the researchers said.

The findings highlight the need to educate clinicians about the appropriate use of antivirals and antibiotics in patients who have symptoms of influenza, the researchers said.

"Continuing education on appropriate antibiotic and antiviral use is essential to improve health care quality," the researchers wrote in their study.

source

It is certainly correct that antibiotics have no effect on influenza and should not be prescribed unless the physician suspects that the person has developed a secondary infection. However, there is increasing evidence that neuraminidase inhibitors such as oseltamivir (Tamiflu) and zanamivir (Relenza) have little benefit for those infected with influenza. Although the proposed mechanism of action and in vitro data appeared very promising (http://biologicalchemistry.tumblr.com/post/71792740352/the-role-of-glycoproteins-in-influenza-infection-and), the clinical data appears to suggest otherwise. A recent meta analysis by the Cochrane Collaboration found that neuraminidase inhibitors reduced the duration of symptoms by only 16.8 hours in adults and had no effect on the development of serious complications. Additionally, oseltamivir was found to increase the rate of nausea and vomiting by approximately 5% and was also found to increase the frequency of psychiatric events compared to placebo. Zanamivir was found to have lower toxicity, likely due to its lower bioavailability. Although it has been suggested that the studies reviewed by the Cochrane Collaboration did not include enough subjects with severe illness or those at high risk of developing complications, the review seems to suggest that the benefits of neuraminidase inhibitors do not significantly outweigh the risks for many patients.

Sources

http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD008965.pub4/abstract;jsessionid=D0952B85723A29FF11B3087DB5F71938.f03t02

— 2 months ago with 151 notes
#science  #medicine  #chemistry  #influenza 
reallymadscientist:

When HIV-1 invades its main target, the immune system’s CD4 T cells, the virus must bind to at least 2 receptors on the cell surface, first to the CD4 glycoprotein and then to either of 2 coreceptors, CCR5 or CXCR4. . 
Recent strategies that are generating promising results involve the use of engineered nucleases that create double-stranded breaks within specified genes. When the cell attempts to repair the induced breaks, mutations are created, and the proteins that are normally made are truncated or not expressed. Paula Cannon, PhD, an associate professor at the University of Southern California, and her team have been using one such nuclease—called a zinc-finger nuclease—to target the CCR5 gene in human hematopoietic stem cells. After they transplanted the altered cells into a “humanized” mouse model (mice engineered to develop a functional human immune system), the human cells retained the ability to differentiate into multiple blood cell lineages that also maintained high rates of CCR5 disruption. When the researchers challenged the transplanted mice with HIV-1, viral replication was blocked and normal levels of human T cells were preserved. “An advantage of this approach is that the stem cells constantly make new CD4 T cells, the natural host cells for HIV, that are effectively resistant to the virus,” said Cannon. “Over time, these cells survive and come to dominate, which allows the host to control the virus.” Cannon and her team are now working to launch a clinical trial.


June and his colleagues have recently completed a clinical trial that uses the same zinc-finger nuclease in a similar strategy in autologous CD4 cells rather than hematopoietic stem cells (Tebas P et al. N Engl J Med. 2014;370[10]:901-910). During the study, 12 patients with chronic HIV infection received 10 billion of their own CD4 cells (11%-28% of which were modified at the CCR5gene) in addition to antiretroviral therapy. One serious adverse event was noted and was attributed to a transfusion reaction. The median CD4 cell count was 1517/μL at week 1, more than 3 times the preinfusion count of 448/μL. An estimated 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 cells were CCR5-modified after 1 week, and the modified cells had an average half-life of 48 weeks. Six of the patients underwent an interruption in antiretroviral treatment 4 weeks after the infusion, and during interruption, the decline in circulating CCR5-modified cells was significantly less than the decline in unmodified cells. HIV RNA even became undetectable in 1 of 4 patients whose RNA could be evaluated, and blood levels of HIV DNA decreased in most patients.




“This is the first example of targeted gene editing in humans, and the work highlights the potential to create an immune system that is resistant to infection by the HIV-1 virus,” said June. The researchers are working to extend their findings to larger numbers of patients and to determine the optimal dose of CCR5-modified T cells.
Article
Image

reallymadscientist:

When HIV-1 invades its main target, the immune system’s CD4 T cells, the virus must bind to at least 2 receptors on the cell surface, first to the CD4 glycoprotein and then to either of 2 coreceptors, CCR5 or CXCR4. . 

Recent strategies that are generating promising results involve the use of engineered nucleases that create double-stranded breaks within specified genes. When the cell attempts to repair the induced breaks, mutations are created, and the proteins that are normally made are truncated or not expressed. Paula Cannon, PhD, an associate professor at the University of Southern California, and her team have been using one such nuclease—called a zinc-finger nuclease—to target the CCR5 gene in human hematopoietic stem cells. After they transplanted the altered cells into a “humanized” mouse model (mice engineered to develop a functional human immune system), the human cells retained the ability to differentiate into multiple blood cell lineages that also maintained high rates of CCR5 disruption. When the researchers challenged the transplanted mice with HIV-1, viral replication was blocked and normal levels of human T cells were preserved. “An advantage of this approach is that the stem cells constantly make new CD4 T cells, the natural host cells for HIV, that are effectively resistant to the virus,” said Cannon. “Over time, these cells survive and come to dominate, which allows the host to control the virus.” Cannon and her team are now working to launch a clinical trial.

June and his colleagues have recently completed a clinical trial that uses the same zinc-finger nuclease in a similar strategy in autologous CD4 cells rather than hematopoietic stem cells (Tebas P et al. N Engl J Med. 2014;370[10]:901-910). During the study, 12 patients with chronic HIV infection received 10 billion of their own CD4 cells (11%-28% of which were modified at the CCR5gene) in addition to antiretroviral therapy. One serious adverse event was noted and was attributed to a transfusion reaction. The median CD4 cell count was 1517/μL at week 1, more than 3 times the preinfusion count of 448/μL. An estimated 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 cells were CCR5-modified after 1 week, and the modified cells had an average half-life of 48 weeks. Six of the patients underwent an interruption in antiretroviral treatment 4 weeks after the infusion, and during interruption, the decline in circulating CCR5-modified cells was significantly less than the decline in unmodified cells. HIV RNA even became undetectable in 1 of 4 patients whose RNA could be evaluated, and blood levels of HIV DNA decreased in most patients.

“This is the first example of targeted gene editing in humans, and the work highlights the potential to create an immune system that is resistant to infection by the HIV-1 virus,” said June. The researchers are working to extend their findings to larger numbers of patients and to determine the optimal dose of CCR5-modified T cells.

Article

Image

(via oxidoreductase)

— 2 months ago with 26 notes
The Dreaded Stereochemistry: Part 1 →

premdstudyforme:

Stereochemistry, for those who may not have taken ochem yet, is the chemistry of isomers. Isomers are molecules with the same chemical formula, but different arrangements. For example, C3H6O can be either 2-propanone (acetone) or 1-propanal. Which look like this:

image

In regards to the Thalidomide anecdote: it is commonly believed that the (R) enantiomer is safe and the (S) enantiomer exhibits teratogenic effects.

image

However, eliminating the teratogenic effects of thalidomide is not so simple as separating the enantiomers and administering only (R)-thalidomide (not that separating enantiomers, especially on an industrial scale, is simple). Thalidomide is rapidly racemized in the body, giving a mixture of the two enantiomers. I believe this occurs via an acid-catalyzed mechanism where protonation of the amide Oxygen promotes deprotonation at the chiral Carbon to give the achiral enolate (it’s an enolate and an enamine at the same time..idk what to call that). The reverse of this reaction then yields a racemic mixture of (R) and (S) thalidomide.

image

Thalidomide is still used, but it is not approved for use in those who are pregnant. According to a Nature Drug Discovery review (link at the bottom), the data supporting the teratogenicity of the S enantiomer over the R are unreliable and other studies have shown the equal teratogenicity of both enantiomers, but I encourage anyone to look at the original studies and make their own claims if they’re so inclined. 

Reference: http://www.nature.com/nrd/journal/v1/n10/box/nrd915_BX1.html

image 1 from http://en.wikipedia.org/wiki/Thalidomide

Mechanism proposed by me 

— 3 months ago with 40 notes
#chemistry  #pharmacology  #pharmacy  #biology  #science 
biologicalchemistry:

454 Pyrosequencing: The Advent of “Next-Generation” Sequencing
Great strides have been made in the last three decades to make DNA sequencing a routine procedure. The Human Genome Project, completed in 2003, used Automated Sanger Sequencing (described in a previous post) to generate a composite sequence of the first human genome for a whopping $2.7 billion over more than a decade. Today, an entire human genome can be sequenced for around $5000 in about a day. This drastic reduction in cost can be linked to the development of so-called next-generation sequencing technologies. The first of these technologies, 454 Pyrosequencing, was commercialized in 2005 by 454 Life Sciences, now owned by Roche. 
The developers of 454 pyrosequencing sought to tackle three problems inherent to first-generation sequencing technologies such as Sanger sequencing: throughput, cost, and library generation. Prior to the development of 454 sequencing, DNA libraries were time-consumingly amplified in vivo, by culturing bacterial cells and isolating clonal DNA from these cells. Additionally, Sanger sequencing has relatively low throughput, yielding a maximum of 1.2 million bases per day. This is only a small fraction of the approximately 3 billion bases in the human genome, drastically limiting the use of Sanger sequencing for large scale sequencing projects. 
454 utilizes a miniaturized version of a previously developed method, pyrosequencing. When a nucleotide is incorporated into the growing DNA strand, a molecule of pyrophosphate is released. The enzyme sulfurylase then converts pyrophosphate to ATP and the enzyme luciferase uses ATP to generate light (A and B above). In 454 pyrosequencing, DNA is amplified by emulsion PCR. PCR (polymerase chain reaction) is a very common technique in molecular biology that uses DNA polymerase to amplify DNA inside of a test tube. Emulsion PCR does the same thing, except  DNA primers are covalently attached to beads and the DNA amplification occurs inside of an oil emulsion. Smaller beads containing the pyrosequencing enzymes are then added to the wells containing the amplified DNA beads (C). Deoxynucleotide triphosphates are then sequentially washed over the plate and light emission is observed. So when A is washed over the plate, light will be emitted from each well where an A is incorporated. When T is washed over the plate, each well where a T is incorporated will emit light. The massively parallel nature of 454 sequencing allows for the generation of significantly more sequence information at a much lower cost per base than Sanger sequencing. The first 454 system generated 20 million bases of sequence information in an 8 hour run with read lengths of 110 bases (significantly less than the 1000 bases per run in Sanger sequencing). Current systems, however, have read lengths approaching 1000 base pairs and can generate over 700 million bases of sequence information in an 8 hour run. 

biologicalchemistry:

454 Pyrosequencing: The Advent of “Next-Generation” Sequencing

Great strides have been made in the last three decades to make DNA sequencing a routine procedure. The Human Genome Project, completed in 2003, used Automated Sanger Sequencing (described in a previous post) to generate a composite sequence of the first human genome for a whopping $2.7 billion over more than a decade. Today, an entire human genome can be sequenced for around $5000 in about a day. This drastic reduction in cost can be linked to the development of so-called next-generation sequencing technologies. The first of these technologies, 454 Pyrosequencing, was commercialized in 2005 by 454 Life Sciences, now owned by Roche. 

The developers of 454 pyrosequencing sought to tackle three problems inherent to first-generation sequencing technologies such as Sanger sequencing: throughput, cost, and library generation. Prior to the development of 454 sequencing, DNA libraries were time-consumingly amplified in vivo, by culturing bacterial cells and isolating clonal DNA from these cells. Additionally, Sanger sequencing has relatively low throughput, yielding a maximum of 1.2 million bases per day. This is only a small fraction of the approximately 3 billion bases in the human genome, drastically limiting the use of Sanger sequencing for large scale sequencing projects. 

454 utilizes a miniaturized version of a previously developed method, pyrosequencing. When a nucleotide is incorporated into the growing DNA strand, a molecule of pyrophosphate is released. The enzyme sulfurylase then converts pyrophosphate to ATP and the enzyme luciferase uses ATP to generate light (A and B above). In 454 pyrosequencing, DNA is amplified by emulsion PCR. PCR (polymerase chain reaction) is a very common technique in molecular biology that uses DNA polymerase to amplify DNA inside of a test tube. Emulsion PCR does the same thing, except  DNA primers are covalently attached to beads and the DNA amplification occurs inside of an oil emulsion. Smaller beads containing the pyrosequencing enzymes are then added to the wells containing the amplified DNA beads (C). Deoxynucleotide triphosphates are then sequentially washed over the plate and light emission is observed. So when A is washed over the plate, light will be emitted from each well where an A is incorporated. When T is washed over the plate, each well where a T is incorporated will emit light. The massively parallel nature of 454 sequencing allows for the generation of significantly more sequence information at a much lower cost per base than Sanger sequencing. The first 454 system generated 20 million bases of sequence information in an 8 hour run with read lengths of 110 bases (significantly less than the 1000 bases per run in Sanger sequencing). Current systems, however, have read lengths approaching 1000 base pairs and can generate over 700 million bases of sequence information in an 8 hour run. 

— 3 months ago with 15 notes
#science  #chemistry  #biology  #DNA 
454 Pyrosequencing: The Advent of “Next-Generation” Sequencing
Great strides have been made in the last three decades to make DNA sequencing a routine procedure. The Human Genome Project, completed in 2003, used Automated Sanger Sequencing (described in a previous post) to generate a composite sequence of the first human genome for a whopping $2.7 billion over more than a decade. Today, an entire human genome can be sequenced for around $5000 in about a day. This drastic reduction in cost can be linked to the development of so-called next-generation sequencing technologies. The first of these technologies, 454 Pyrosequencing, was commercialized in 2005 by 454 Life Sciences, now owned by Roche. 
The developers of 454 pyrosequencing sought to tackle three problems inherent to first-generation sequencing technologies such as Sanger sequencing: throughput, cost, and library generation. Prior to the development of 454 sequencing, DNA libraries were time-consumingly amplified in vivo, by culturing bacterial cells and isolating clonal DNA from these cells. Additionally, Sanger sequencing has relatively low throughput, yielding a maximum of 1.2 million bases per day. This is only a small fraction of the approximately 3 billion bases in the human genome, drastically limiting the use of Sanger sequencing for large scale sequencing projects. 
454 utilizes a miniaturized version of a previously developed method, pyrosequencing. When a nucleotide is incorporated into the growing DNA strand, a molecule of pyrophosphate is released. The enzyme sulfurylase then converts pyrophosphate to ATP and the enzyme luciferase uses ATP to generate light (A and B above). In 454 pyrosequencing, DNA is amplified by emulsion PCR. PCR (polymerase chain reaction) is a very common technique in molecular biology that uses DNA polymerase to amplify DNA inside of a test tube. Emulsion PCR does the same thing, except  DNA primers are covalently attached to beads and the DNA amplification occurs inside of an oil emulsion. Smaller beads containing the pyrosequencing enzymes are then added to the wells containing the amplified DNA beads (C). Deoxynucleotide triphosphates are then sequentially washed over the plate and light emission is observed. So when A is washed over the plate, light will be emitted from each well where an A is incorporated. When T is washed over the plate, each well where a T is incorporated will emit light. The massively parallel nature of 454 sequencing allows for the generation of significantly more sequence information at a much lower cost per base than Sanger sequencing. The first 454 system generated 20 million bases of sequence information in an 8 hour run with read lengths of 110 bases (significantly less than the 1000 bases per run in Sanger sequencing). Current systems, however, have read lengths approaching 1000 base pairs and can generate over 700 million bases of sequence information in an 8 hour run. 

454 Pyrosequencing: The Advent of “Next-Generation” Sequencing

Great strides have been made in the last three decades to make DNA sequencing a routine procedure. The Human Genome Project, completed in 2003, used Automated Sanger Sequencing (described in a previous post) to generate a composite sequence of the first human genome for a whopping $2.7 billion over more than a decade. Today, an entire human genome can be sequenced for around $5000 in about a day. This drastic reduction in cost can be linked to the development of so-called next-generation sequencing technologies. The first of these technologies, 454 Pyrosequencing, was commercialized in 2005 by 454 Life Sciences, now owned by Roche. 

The developers of 454 pyrosequencing sought to tackle three problems inherent to first-generation sequencing technologies such as Sanger sequencing: throughput, cost, and library generation. Prior to the development of 454 sequencing, DNA libraries were time-consumingly amplified in vivo, by culturing bacterial cells and isolating clonal DNA from these cells. Additionally, Sanger sequencing has relatively low throughput, yielding a maximum of 1.2 million bases per day. This is only a small fraction of the approximately 3 billion bases in the human genome, drastically limiting the use of Sanger sequencing for large scale sequencing projects. 

454 utilizes a miniaturized version of a previously developed method, pyrosequencing. When a nucleotide is incorporated into the growing DNA strand, a molecule of pyrophosphate is released. The enzyme sulfurylase then converts pyrophosphate to ATP and the enzyme luciferase uses ATP to generate light (A and B above). In 454 pyrosequencing, DNA is amplified by emulsion PCR. PCR (polymerase chain reaction) is a very common technique in molecular biology that uses DNA polymerase to amplify DNA inside of a test tube. Emulsion PCR does the same thing, except  DNA primers are covalently attached to beads and the DNA amplification occurs inside of an oil emulsion. Smaller beads containing the pyrosequencing enzymes are then added to the wells containing the amplified DNA beads (C). Deoxynucleotide triphosphates are then sequentially washed over the plate and light emission is observed. So when A is washed over the plate, light will be emitted from each well where an A is incorporated. When T is washed over the plate, each well where a T is incorporated will emit light. The massively parallel nature of 454 sequencing allows for the generation of significantly more sequence information at a much lower cost per base than Sanger sequencing. The first 454 system generated 20 million bases of sequence information in an 8 hour run with read lengths of 110 bases (significantly less than the 1000 bases per run in Sanger sequencing). Current systems, however, have read lengths approaching 1000 base pairs and can generate over 700 million bases of sequence information in an 8 hour run. 

— 3 months ago with 15 notes
#science  #biology  #chemistry  #DNA  #DNA sequencing  #biochemistry