Tuesday, January 28, 2020
Cell-Permeable Stapled Peptides from HIV1 Gene Products
Cell-Permeable Stapled Peptides from HIV1 Gene Products Cell-Permeable Stapled Peptides Based on HIV-1 Integrase Inhibitors Derived from HIV-1 Gene Products Introduction HIV-1 Integrase (IN), is necessary enzyme for the replication of the virus into the host cells. It catalyzes the integration of viral DNA into the genome of host cells. The mechanisms employed by the enzyme are strand transfer and 3â⬠²-end processing reactions. The enzyme is vital for the virus life cycle and as a result it is a target for anti-HIV-1 drugs. It is reported from previous studies that HIV-1 protein Vpr has inhibitory effect on Integrase activity. The inhibition is a result of protein-protein interactions. Specifically Vpr interacts through its C- terminal domain. Results from previous studies indicate that the active petptide inhibition motif is peptide 1 (Figure 1). Figure 1. Amino acid sequences of peptides 1 and 2, and outline of the study. Regarding the SAR studies, E-K pairs were introduced in i and i + 4 positions to increase the helicity, by forming salt bridges. In the same study to peptide 1 was added an octa-arginine tail, the purpose of which was to improve, cell permeability. The presence of the octa-arginine tail however resulted in increase of cytotoxicity of the peptide. The hypothesis on which the current study was based is that by finding a different way to improve cell permeability without the 8-R tail the cytotoxicity of the peptide would decrease. The idea was to make the peptide more lipophilic, by adding hydrophobic groups on the external of the a-helix, and at the same time improve its helicity. An available method to achieve that is the use of a-helix stabilized peptidomimetics. Specifically, hydrocarbon stapled peptides, in which a hydrocarbon link is formed between successive turns, via a ring closed metathesis (RCM) reaction, were used. By having a hydrocarbon linkage between positions i and i+4 in the a-helix, the helicity is improved because the two positions are covalently bound via the hydrocarbon chain, and simultaneously the lipophilicity and cell permeability of the peptide is increased because of the presence of a hydrophobic hydrocarbon chain on the external part of the a helical structure. In Figure 1 is presented an outline of the study. Design and synthesis of the peptides The lead peptide sequence used was sequence 1 (Fig.1). From previous studies it was known that residues: Phe12, Ile13, Phe15, and Ile17 are vital for IN inhibition activity. Stapling was performed between i and i+4 positions by avoiding substitution of these residues. Protected linear peptides were constructed by Fmoc-solid phase peptide synthesis. The RCM reactions of the protected linear peptides were performed on the resin by treatment with ruthenium-mediated Grubbs second catalyst in 1,2-dichloroethane. All hydrocarbon and all ether stapling was performed, however the first technique resulted in higher yields as itââ¬â¢s presented in Table 1. After RCM, the peptides were deprotected, acetylated, and cleaved from the resin. Purification of the stapled peptides was done by HPLC. However it is not the purpose of this paper to present experimental details. Table 1. Sequences of Designed Stapled Peptides and Their Conversion Yields of RCM Reactions CD Spectroscopy of Linear and Stapled Peptides. CD Spectroscopy was used to analyze the secondary structures of the synthetic stapled peptides, 3SâËâ9S and 11S. Linear peptides, 4LâËâ6L, 8L, 9L, and 11L, were used as control (Figure 3). Negative peaks at 208 and 222 nm, are characteristic for à ±-helical structures. All stapled peptides, except 3S, formed à ±-helices. The corresponding linear peptides on the other hand including compound 1 showed a broad negative peak around 215 nm, which indicates a à ²-sheet structure. It is obvious that stapling of the linear peptides leads to the formation of à ±-helices. Figure 3. CD Spectra of stapled peptides (left) and corresponding linear (right). Integrase (IN) Inhibition Assays and MT-4 Luc Assays (anti HIV). The next step was to determine the potency of the synthesized peptides using integrase inhibition assays. IC50 values were determined for stapled and linear compounds regarding both 3-end processing and strand transferring reactions (Figure 4 Left). Anti HIV activity was also screened for all peptides using MT-4 Luc Assays (Figure 4 Right). Figure 4. IC50 Values (Left). MT-4 Luc Assay (Right) All compounds had integrase inhibition activity, however lead compound 2 was way more potent. Regarding the MT-4 Luc assays compound 6S showed similar anti HIV activity as lead compound 2 at concentrations higher than 2.5 um. Compound 8S also showed significant activity at concentrations higher than 5 um. 6S and 2 have almost the same level of anti-HIV activity in cells, however 2 has much higher IN inhibitory activity in vitro than compound 6S. Imaging Experiments Stapled and linear peptides were labeled with fluorescein and imaging experiments were performed to examine their cell permeability. The stapled peptides 6S and 8S demonstrated improved cell permeability compared to the lead compound and the linear peptides. Octaarginine tail functional role investigation. The next step was to study the functional role of the (Arg) 8 tail, besides influencing cell permeability. By using 6S and 6L as lead compounds, a new set of peptides was synthesized (Figure 5). Specifically conjugates of 6S with octaarginine (17) and with the quartet repeat of arginine and glutamic acid (18). For 6L, with the quartet repeat of arginine and glutamic acid (19), and the conjugates of 6S with tetra-arginine (20), penta-arginine (21), hexa-arginine (22), and hepta-arginine (23). IC50 values were determined for the new set of peptides and also MT-4 Luc assays were performed. The presence of the octaarginine tail is necessary for the inhibitory activity however it still makes the peptides cytotoxic. Different lengths in the Arg tail were also examined. Still the octaarginine (17) conjugated peptide is the most potent but compounds 22 and 23 had good potency and lower cytotoxicity than 17, so those compounds were selected as lead compounds for further development. DNA binding experiments were also performed to examine the role of Arg8 tail, and the results indicate that 17 has high DNA binding affinity and that might influence the high inhibition activity of the peptide. Conclusions and Critical Review Stapling caused a significant increase in à ±-helicity and cell membrane penetration, and in the expression of potent anti-HIV activity in cells. Stapling and the addition of octa-arginine caused cell membrane penetration. Stapling by itself did not involve cytotoxicity while incorporation of octa-arginine into the structures increased the cytotoxicity of the compounds. Generally it was a well-organized paper. It had good flow of information and background information was provided. The hypotheses were not stated but were easy to understand. The experiments and the data supported the conclusions and the concepts. The figures and the graphs were interpretable and accurate in supporting the conclusions related to them. However there are several interesting points to be addressed. The first point is the configuration of the new double bond formed. It would be interesting to see if the configuration of the double bond would affect the secondary structure and the helicity of the peptides. Another question that need to be addressed is why the stapled peptide 3S did fold into b sheet structure unlike the other stapled peptides which formed a helices. It would be interesting to examine the role of the substituted amino acids of 3S, in the folding of the peptide. Regarding the role of the octaarginine tail, the DNA binding experiments indicate that the octaarginine conjugated peptide has affinity for DNA binding. However it is unclear how exactly the tail affects the potency and the cytotoxicity. Further experiments are necessary to clarify that. I would suggest some NMR experiments to investigate the binding. First 1H-15N HSQC may be performed to the (Arg)8 fused peptide before and after binding with DNA. That would show which residues are involved in binding and would give data to determine the specific interaction. References: Nomura, Wataru, et al. Cell-Permeable Stapled Peptides Based on HIV-1 Integrase Inhibitors Derived from HIV-1 Gene Products. ACS chemical biology 8.10 (2013): 2235-2244.
Monday, January 20, 2020
Health Insurance Portability and Accountability Act Essay -- Healthcar
The Standards for Privacy of Individually Identifiable Health Information, better known as the Privacy Rule, that took effect in April 2003 for large entities and a year later for small ones, was established as the first set of national standards for the protection of health information. This rule was issued by the U.S. Department of Health and Human Services to meet the requirement of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). The Privacy Rule was born out of a need for health information to be appropriately protected yet still allowing the health information to be shared to ensure quality health care and to protect the publicââ¬â¢s health and well being. It allows for the protection of the privacy of the patient and yet it also permits vital uses of information. The Privacy Rule established a category of health information that a covered entity may only use or disclose to others in certain circumstances and under certain conditions. According to the National Institute of Health Publication Number 03-5388, the protected information is known as Protected Health Information (PHI) and includes individually identifiable health information transmitted by electronic media, maintained in electronic media, or transmitted or maintained in any other form or medium. However, it excludes education records covered by the Family Educational Rights and Privacy Act, as amended, 20 U.S.C. 1232g, records described at 20 U.S.C. 1232g(a)(4)(B)(iv), and employment records held by a covered entity in its role as employer. Covered entities are organizations that are subject to the Privacy Rule. Three types of covered entities are 1) Health Plans - which are individual or group plans that provide or pay the cost of medi... ... and HIPAA, Does instant access and availability from mobile technology jeopardize patient privacy? [Electronic version] Nursing Management, June 2007, 38-40 Martin, J. (2008). Whoââ¬â¢s looking at your medical records? COPDConnection.com Retrieved November 12, 2011, from http://www.healthcentral.com/copd/c/19257/30481/comments Robertson, L. (2008). Whoââ¬â¢s looking at your medical records? [Electronic version] Saturday Evening Post, 280.3, 54-92, Consumer Health Complete. Protecting Personal Health Information in Research: Understanding the HIPAA Privacy Rule, NIH Publication Number 03-5388. Retrieved November 12, 2011 from http://privacyruleandresearch.nih.gov/pr_02.asp Summary of the HIPAA Privacy Rule, Office of Civil Rights Privacy Rule Summary, (2003). Retrieved November 12, 2011 from http://www.hhs.gov/ocr/privacy/hipaa/understanding/summary/privacysummary.pdf
Sunday, January 12, 2020
Word Narrative
Collaborative practice I've been in hospital a few days now. I've settled in. Well as much as you can be expected to when you're in a strange environment. The first day was awful for me though, I had no idea what was going on. I'd just started my 3 weeks annual leave (l work abroad so I was looking forward to spending time with my family etc. ) and I started having severe chest pains at home. They were crushing pains I can't explain the pain in any other way than it felt like a vice. My wife called 999 and the ambulance asked a lot of questions, before I knew t I was on my way to hospital.I remember everyone just running around, it felt like no one was actually talking to me. Like I was invisible! The doctors were saying something about a possible stroke, but not to me, they were talking amongst themselves. They attached a load of wires to me and the monitor they attached me to constantly beeping. Still drives me crazy that noise all around the ward. Some of the nurses are nice, not all of them seem to like me though. Maybe I'm an inconvenience? Hope they don't think that. I know they are all really busy, that's obvious.I finally found out that have suffered a stroke and I need to stay in the hospital until I'm well enough to go home. Although most of what they said to me was not in a language I could understand! I can remember asking where my wife was a lot and no one answering me, which was really frustrating as I knew she would be equally as worried. Didn't get told when would be able to see her for at least an hour after arriving at the hospital. The ward I'm on has really strict visiting hours so my wife can only visit me for two hours a day and she isn't allowed to bring he children which is a shame.But have been told I will probably only spend 5 days on this ward and that if I'm not ready to go home I will be moved on to a ward with more flexible visiting hours. I'm looking forward to that. The thing that hate the most about being on a ward is having to use a commode behind a curtain. I am a 41 year old man and to be honest I find it very embarrassing and undignified. I'm not saying my experience her has been completely unpleasant but the sooner I get out of here the better and I will be in no hurry to come back.
Friday, January 3, 2020
Rape Trauma Syndrome Essays - 1705 Words
You can most commonly find dictionaries defining rape as a sexual act committed by force especially on a woman (American Heritage). Until a few years ago it was limited to penile penetration of the vagina. Penal Code two hundred sixty-one defines rape as an act of sexual intercourse accomplished with a person who is not the spouse of the perpetrator without the lawful consent (Roberson). Penal Code two hundred sixty-three goes on to say that the fundamental wrong at which the law of rape is aimed is . . . the violation of a womans will and sexuality (Roberson). All other sexual assaults are categorized under different names, yet the result is most often the same. To most Americans Rape has a tendency to be one of the cruelestâ⬠¦show more contentâ⬠¦These are devastating losses. Thus, many survivors report that the offender irrevocably changed their lives. The offender steals a part of the victim that can never be returned. We can examine the impact of rape in various ways. One perspective takes into consideration the underlying meaning the assault has for the victim. The grave threats and incredible losses the victim suffers alters their life dramatically, sometimes forever. Another manifestation of the impact of rape is the anguish of the victims. The psychological and behavioral symptoms of distress are poignant symbols of the destructiveness of the offenderââ¬â¢s acts. Yet another way to understand the impact of rape is to compare it with other forms of trauma. Rape is an extreme form of human cruelty that in some ways mirrors other overwhelming traumatic events. Rape Trauma Syndrome is the acute phase and long-term reorganization process that happens because of forcible rape or attempted forcible rape (American Journal). Researchers have discovered stages that the victim goes through while suffering the effects of victimization (Parad). The first impact of rape is a feeling of numbness; the victim is in a state of shock and disbelief and still may be in terror (American Journal). The victims appearance can be misleading, appearing jolted or astonished, sedate and stifled (Parad). The controlled behavior covers up the serious psychological wound they are suffering from (Parad). Expressions of disbelief are mostShow MoreRelatedEssay about Psychological Effects of Sexual Assault1504 Words à |à 7 Pagesinterchangeably with the word rape. The decision on whether or not to use the term rape or sexual assault is made by a stateââ¬â¢s jurisdiction. Sexual assault is more readily used in an attempt to be more gender neutral (National Victim Center). Sexual assault can be most easily described as forced or unconsentual sexual intercourse. The individual that is performing these acts on the victim may ei ther be a stranger or an acquaintance. 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This paper will evaluate rape, as well as the effects it has on women, the theory behind male dominance and patriarchyRead MoreMilitary Sexual Trauma ( Mst ) Essay1135 Words à |à 5 PagesMilitary Sexual Trauma (MST) Throughout many wars that the United States of America had endured within the 238 years, recently America have another war to handle which is Military Sexual Trauma. Only recently the social media decided to take part of acknowledging that many veterans have mental health issues. However, they are mainly focusing on one problem which is Post Traumatic Stress Disorder (PTSD). 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When a woman gets raped, she experiences Rape Trauma Syndrome (a medical term given to the response that survivors of rape). If the incident caused the woman to get pregnant, there is a high possibility that she might want to get an abortion. Sam Sherwin writes ââ¬Å"If the fetus is the result of rape or incest, then the psychological pain of carrying it may be unbearableRead MoreHuman Trafficking Is A Problem Within The U.s. Essay1617 Words à |à 7 Pageslocal law enforcement officials. Raids as an anti-sex trafficking measure severely impact sex trafficked victims. Because of their complicated legal status and their language barriers, the arrest or fear of arrest creates stress and other emotional trauma for trafficking victims. Victims may also experience physical violence from law enforcement during raids. Trafficking victims are also exposed to different psychological stressors. They suffer social alienation in the host and home countries. 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It can also be anything that forces a person to be in unwanted sexual contact. There are many examples of sexual abuse like voyeurism, exhibitionism, incest, and sexual harassment. Voyeurism
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