PSAB ACTIVITIES
- ASM Presents Comments to
FDA/CDRH at Public Meeting on Oversight of Laboratory Developed Tests
- ASM Presents Comments at CMS
Meeting on Coding for New Clinical Lab Tests
- ASM Comments on the WMD
Prevention and Preparedness Act of 2010
- ASM
Comments for the Trans Atlantic Task Force on Antimicrobial Resistance
ASM NEWS, JOURNAL ARTICLES AND
UPDATES
- ASM ICAAC Meeting
- ASM 2011 General Meeting Awards
- ASM Upcoming Audioconferences
- ASM Journal Articles of
Interest
FEDERAL AGENCY UPDATES
- CDC
Health Advisory on Increased Potential for Dengue Infection in Travelers
Returning from International and Selected Domestic Areas
- MMWR Articles of Interest
- First
Smallpox Vaccine for Special Populations Delivered Under Project BioShield
- Technology Assessment for Quality, Regulation
and Clinical Utility of LDTs
- NIH
Scientists Advance Universal Flu Vaccine
- National
HIV/AIDS Strategy
OTHER INFORMATION AND UPDATES
- New from CLSI
- NLTN Online Training Course in
Packing and Shipping
- Articles of Interest
ASM Presents Comments to FDA/CDRH at
Public Meeting on Oversight of Laboratory Developed Tests
On July 20,
the ASM presented comments at a public meeting sponsored by the Food and Drug
Administration’s (FDA) Center for Devices and Radiological Health (CDRH) on
Oversight of Laboratory Developed Tests (LDTs): Clinical Lab Challenges. The purpose of the meeting was to provide a
forum where interested stakeholders could present comments regarding reasonable
and effective regulation of LDTs. The
meeting took place in Hyattsville,
MD on July 19-20, 2010 and
Laboratory Practices Committee Member, Paula Revell,
represented ASM at the meeting and presented ASM’s comments. To read ASM’s statement, go to http://www.asm.org/index.php/policy/ldtcomments7-20-10.html
ASM Presents Comments at CMS Meeting
on Coding for New Clinical Lab Tests
Vickie
Baselski, Chair of the Professional Affairs Committee presented ASM comments to
the Centers for Medicare & Medicaid Services (CMS) on new clinical
laboratory tests that will be included on the 2011 Medicare Clinical Laboratory
Fee Schedule. To read ASM’s comments, go
to http://www.asm.org/index.php/policy/cmspaymentcomments-2010.html
ASM Comments on the WMD Prevention
and Preparedness Act of 2010
ASM sent
comments to the House Committee on Homeland Security commenting on H.R. 5498,
the WMD Prevention and Preparedness Act of 2010. To read ASM’s comments, go to http://www.asm.org/index.php/policy/wmdactof2010-comments.html
ASM
Comments for the Trans Atlantic Task Force on Antimicrobial Resistance
On June 7,
the ASM presented comments to the Department of Health and Human Services (HHS)
at the Stakeholder Listening Session convened to discuss the work of the
Trans-Atlantic Task Force on Antimicrobial Resistance (TATFAR). The ASM commended the establishment of the
TATFAR, provided some preliminary comments on addressing antimicrobial
resistance and requested more information about how it could assist the Task
Force as it pursues its focus on urgent antimicrobial issues world wide. The
TATFAR, which is a government to government task force, was formed following
the November 3, 2009 US-European Union Summit Declaration which focused on the
specific areas of appropriate therapeutic use of antimicrobial drugs in the
medical and veterinary communities, prevention of both healthcare and community
associated drug resistant infections and strategies for improving the pipeline
of new antimicrobial drugs.
The ASM
comments are available at http://www.asm.org/index.php?option=com_content&view=article&id=91489.
ASM NEWS, JOURNAL ARTICLES AND
UPDATES
ASM ICAAC Meeting
The 2010 Interscience Conference on
Antimicrobial Agents and Chemotherapy (ICAAC) meeting will be celebrating its
50th year in Boston,
MA. The meeting will be held September
12-15. For more information, visit the
ICAAC website at http://www.icaac.org/
ASM 2011 General Meeting Awards
Nominations
are being accepted for ASM’s 2011 General Meeting Awards. The awards honor leadership, research and
service. The nomination deadline is
October 1. The 2011 General Meeting will
be held in New Orleans, LA May 21- 24, 2011. To submit a nomination, go to http://www.asm.org/awards
ASM Upcoming Audioconferences
Pertussis in the 21st
Century: The Laboratory Diagnostic Challenge Continues
Date:
August 11, 2010
Time: 1:00 PM, Eastern Time
Speaker: Mario Marcon
Pros and Cons of Screening Assays in
the Diagnosis of Tuberculosis
Date: August 25, 2010
Time: 1:00 PM, Eastern Time
Speaker: Rebecca Horvat
For more
information including registration, go to http://www.asmaudio.org/teleconference.asp#sched
ASM Journal Articles of Interest
Quitting
Smoking May Minimize Harmful Bacteria and Replenish Health Bacteria
Journal
of Clinical Microbiology, July 2010
Microbicide
Containing Engineered Bacteria May Inhibit HIV-1
Antimicrobial
Agents and Chemotherapy, July 2010
Salmonella
Contaminated Pork May Pose Health Risk for Humans
Applied
and Environmental Microbiology, July 2010
Prior
Exposure to Seasonal Influenza May Explain the Mildness of the 2009 H1N1
Pandemic
Journal
of Virology, July 2010
For as
summary of the articles and to download abstracts, go to http://www.asm.org/index.php/news-room/journal-tipsheets.html
FEDERAL AGENCY UPDATES
CDC
Health Advisory on Increased Potential for Dengue Infection in Travelers
Returning from International and Selected Domestic Areas
The Centers for Disease Control and Prevention (CDC) sent a Health
Advisory to clinicians regarding the increased potential for dengue infection in
travelers returning from the many parts of the tropics and subtropics. Dengue virus transmission has been increasing to epidemic
levels in many parts of the tropics and subtropics. Travelers to these areas
are at risk of acquiring dengue virus and developing dengue fever (DF) or the
severe form of the disease, dengue hemorrhagic fever (DHF). CDC strongly advises that health care
providers in the United States should: 1) consider DF and DHF when evaluating
patients returning from dengue-affected areas, both domestic and abroad, who
present with an acute febrile illness within two weeks of their return, 2)
submit serum specimens for appropriate laboratory testing, and 3) report all
presumptive and confirmed cases of DF and DHF to their local or state health
department. To read the Health Advisory,
go to http://www2a.cdc.gov/HAN/ArchiveSys/ViewMsgV.asp?AlertNum=00315
MMWR Articles of Interest
Emergence of Cryptococcus
gattii, Pacific Northwest, 2004 - 2010
July 23, 2010 / 59(28);865-868
La Crosse Virus Neuroinvasive
Disease, Missouri, 2009
July 23, 2010 / 59(28);869-871
Gastrointestinal Anthrax after
an Animal-Hide Drumming Event, New Hampshire
and Massachusetts,
2009
July 23, 2010 / 59(28);872-877
Notes from the Field: Dengue
Epidemic, Puerto Rico, January - July 2010
July 23, 2010 / 59(28);878
Use of Anthrax Vaccine in the United States: Recommendations of
the Advisory Committee on Immunization Practices (ACIP), 2009
July 23, 2010 / 59(rr06);1-30
West Nile Virus Activity, United States,
2009
July 2, 2010 / 59(25);769-772
First
Smallpox Vaccine for Special Populations Delivered Under Project BioShield
Delivery to
the Strategic National Stockpile of the first 1 million doses of the nation’s
first smallpox vaccine for certain immune compromised populations is now
complete as the result of a Project BioShield contract. Under this contract,
the Danish company Bavarian Nordic is manufacturing and delivering 20 million
doses of its next generation smallpox vaccine known as modified vaccinia Ankara
(MVA) or Imvamune. In an emergency, such
as the virus being obtained from a secure lab and used in an act of terrorism,
the vaccine may be authorized for use to protect people who have weakened
immune systems, specifically HIV persons who have not progressed to AIDS. For
more information, go to http://www.hhs.gov/news/press/2010pres/07/20100714c.html
Technology Assessment for Quality, Regulation and
Clinical Utility of LDTs
On May 19, Technology Assessment for Quality, Regulation and Clinical
Utility of Laboratory Developed Molecular Tests was published by the ECRI
Institute Evidence
Based Practice
Center under contract for
the Agency for Health Research and
Quality. The report, which was requested
by the Coverage and Analysis Group at the Centers for Medicare &
Medicaid Services, summarizes the available scientific evidence on the quality
of laboratory developed tests (LDTs) that are not regulated by the Food and Drug
Administration. The report includes
information on the 1) types of LDTs available for conditions relevant to the
Medicare population; 2) methodologies and processes that have been developed
for the assessment of analytical and clinical performance of molecular tests,
3) summarizes the role of Federal agencies in regulating LDTs, and 4)
identifies the quality standards that have been developed for molecular tests
by regulatory bodies, industry and the medical community. The report can be downloaded at http://www.cms.gov/determinationprocess/downloads/id72TA.pdf
NIH
Scientists Advance Universal Flu Vaccine
A universal
influenza vaccine may become a reality because of research led by scientists
from the National Institute of Allergy and Infectious Diseases (NIAID), part of
the National Institutes of Health. In
experiments with mice, ferrets and monkeys, the investigators used a two-step
immunization approach to elicit infection fighting antibodies that attacked a
diverse array of influenza virus strains. Current flu vaccines do not generate
such broadly neutralizing antibodies, so they must be reformulated annually to
match the predominant virus strains circulating each year. For more information, go to
http://www.nih.gov/news/health/jul2010/niaid-15.htm
National
HIV/AIDS Strategy
On July 13,
President Obama released the new National HIV/AIDS Strategy. The strategy is an across-the-board approach
that calls for a more coordinated national response to achieve three primary,
concrete goals: (1) reduce new HIV
infections, (2) increase access to care and optimize health outcomes for people
with HIV/AIDS, and (3) reduce HIV-related health disparities. For more information, go to http://aids.gov/federal-resources/policies/national-hiv-aids-strategy/
OTHER INFORMATION AND UPDATES
New from CLSI
M100-S20 June 2010 Update, Performance Standards for Antimicrobial
Susceptibility Testing; Update (M100-S20-U)
To purchase
this and other CLSI products, go to http://www.clsi.org
NLTN Online Training Course in
Packing and Shipping
The
National Laboratory Training Network (NLTN) is sponsoring an interactive online
training course, “Packaging &
Shipping Division 6.2 Materials.”
The intermediate level online course is designed for individual study
and is suitable for those seeking recertification. Participants are provided with information
useful for complying with regulations through use of instructional content and
the opportunity to apply knowledge using realistic scenarios. For more information, go to http://www.nltn.org/302-10.htm
Articles of Interest
To
fight drug-resistant MRSA, algorithm predicts bacteria’s future mutations
Popular
Science, July 20, 2010
Researchers
led by Bruce Donald, a professor of computer science and biochemistry at Duke University,
are using a computer algorithm to predict MRSA’s next move.
Enlist
malaria-resistant mosquitoes to stop its spread
New
Scientist, July 20, 2010
Michael
Riehle at the University of Arizona at Tucson and
colleagues put a novel gene into the mosquito species that carries malaria in India. The new
gene permanently switched on a set of genes normally affected by insulin and
involved in the immune system.
Synthetic
cell-like microcapsules communicate like biological cells, cooperate like ants
Popular
Science, July 20, 2010
Taking cues
from slime molds, ants, and living biological cells, a team of University of Pittsburgh researchers has designed a
system of artificial cells that can communicate with one another and cooperate
to carry out tasks. The computer models they have devised could lead to
artificial cellular systems that perform highly specialized jobs at the
microscopic level.
This
won't hurt a bit
Science
News, July 18, 2010
Researchers
have invented a new vaccine-delivery system that replaces the large single
needle with 100 tiny dissolvable ones embedded in a Band-Aid–like patch. The
new patch can immunize mice against influenza just as effectively as
conventional needle vaccination, its developers report online July 18 in Nature Medicine.
Retrovirus replication process different than thought
Science
Blog, July 15, 2010
How a
retrovirus, like HIV, reproduces and assembles new viruses is different than
previously thought, according to Penn State College of Medicine researchers.
Understanding the steps a virus takes for assembly could allow development of a
way to prevent the spread of retroviral diseases.
Few
people cover coughs and sneezes
Reuters, July
13, 2010
Lots of
people aren't getting the message about covering their coughs and sneezes, at
least in New Zealand,
according to a study conducted during last year's H1N1 swine flu pandemic.
Study
suggests link between HPV, skin cancer
U.S. News
& World Report, July 8, 2010
The
ubiquitous virus linked to cervical, vaginal and throat cancers may also raise
the risk of developing squamous cell carcinoma, the second most common form of
skin cancer, a new study suggests.
Amid
the murk of 'gut flora,' vitamin D receptor emerges as a key player
Science
Daily, July 8, 2010
Scientists
have found that the vitamin D receptor is a key player amid the gut bacteria, what
scientists refer to matter-of-factly as the "gut flora, "helping to
govern their activity, responding to their cues, and sometimes countering their
presence. The findings deliver a new lead to scientists investigating how
bacteria might play a role in the development of inflammatory bowel diseases
such as Crohn's disease or ulcerative colitis.
Surgery
linked to Creutzfeldt-Jakob disease
Science
Blog, July 8, 2010
The finding
reveals that, with a few exceptions, the risk of having contracted the sporadic
form of CJD manifests itself at least 20 years after having undergone an
operation.
Souped-up
antibody fends off HIV
Nature,
July 8, 2010
An antibody
that can block more than 90% of strains of HIV-1, the most common form of the
disease, has been discovered using a method for fishing specific proteins out
of the blood.
Map
of herpes virus protein suggests a new drug therapy
Newswise, July
6, 2010
New
research reveals the unusual structure of the protein complex that allows a
herpes virus to invade cells. This detailed map of a key piece of the herpes
virus “cell-entry machinery” gives scientists a new target for antiviral drugs.
Revolutionary
medical dressing uses nanotechnology to fight infection
Science
Daily, July 6, 2010
Researchers
are using nanotechnology to develop a medical dressing which will detect and
treat infection in wounds.
Bacterial
communication encourages chronic, resistant ear infections
Science
Blog, July 6, 2010
Ear
infections caused by more than one species of bacteria could be more persistent
and antibiotic-resistant because one pathogen may be communicating with the
other, encouraging it to bolster its defenses. Researchers from Wake Forest
University Baptist
MedicalCenter
publish their findings today in mBio,
the online open-access journal of the American Society for Microbiology
Vaccine for Marburg
virus passes monkey test
Science News, July 5, 2010
A devastating tropical virus that has no cure can be
ambushed by vaccination a day or two after exposure, tests in monkeys show.
Discovery of Controlled Swarm in Bacteria: Could Help
Design New Strategies to Increase Sensitivity to Antibiotics
ScienceDaily, June 30, 2010
A study led by researchers from Universitat Autònoma de
Barcelona (UAB) describes one of the mechanisms in which pathogenic bacteria
populations control the way they spread over the surface of the organs they
infect and stop when they detect the presence of an antibiotic, only to resume
again when the effect wears off. The star of this process is the RecA protein,
which significantly increases its concentration at the start of the bacteria
DNA repair mechanism induced by antibiotics.
Deaths in the family cause bacteria to flee
PhysOrg, June 29, 2010
The deaths of nearby relatives has a curious effect on the
bacterium Caulobacter crescentus, surviving
cells lose their stickiness. Indiana University Bloomington biologists report
in an upcoming issue of Molecular Microbiology that exposure to the
extracellular DNA (eDNA) released by dying neighbors stops the sticky holdfasts
of living Caulobacter from adhering to surfaces, preventing cells from joining
bacterial biofilms. Less sticky cells are more likely to escape established
colonies, out to where conditions may be better.
For additional information about any
items in this newsletter, please contact Suzy Leous, Manager, Public Affairs,
ASM, at sleous@asmusa.org.
Conclusions
In conclusion, this study demonstrates that h-SH3 can inhibit the replication of influenza A viruses (H1N1, H3N2, and H9N2) at different degrees, and targeting NS1-p85β interaction (PI3K/Akt pathway) or other virus-host interaction may be an attractive strategy against the infection of various influenza A virus subtypes/strains.
Methods
Cell lines, viruses, plasmids, and reagents
Madin-Darby canine kidney (MDCK) cell lines were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and antibiotics (100 U penicillin and 100 ug/ml streptomycin) at 37℃ in 5% CO2.
Influenza A virus strains A/PR/8/34(H1N1), A/ST/1233/2006(H1N1), A/ST/364/2005(H3N2), A/Ph/ST/2246/2006(H9N2), and A/Qa/ST/199/2006(H9N2), abbreviated herein to PR8, ST1233, ST364, Ph2246, and Qa199, respectively, were used in this study.
SH3 domain (9-79aa) of p85β subunit was cloned into PNF vector (a modified pcDNA3 plasmid with an N-terminal Flag tag) at the BamHI and EcoRI sites to generate pSH3. Full-length NS1 sequence of H1N1, H3N2, or H9N2 viruses was cloned into pEGFP-c1 at the BamHI site to generate pNS11, pNS32, or pNS92 plasmids, respectively. The constructs were verified by DNA sequencing.
Rabbit monoclonal anti-phospho-Akt (Ser473) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA), Rabbit monoclonal anti-Flag antibody from Sigma (St. Louis, MO, USA), Rabbit monoclonal anti-Akt antibody, Cy3-labeled goat anti-rabbit antibody, peroxidase-conjugated goat anti-rabbit antibody, and LY294002 (PI3K inhibitor) from Beyotime Biotechnology (Jiangsu, China), and Lipofectamine 2000 from Invitrogen (Carlsbad, CA, USA).
Co-localization analysis by confocal microscopy
MDCK cell cultures on the glass coverslips were transfected with indicated plasmids. 24 h post-transfection, the cells were washed twice with phosphate-buffered saline (PBS), fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 7 min, and incubated for 30 min in PBS containing 3% Bovine Serum Albumin (BSA). Cells were subsequently incubated at room temperature with anti-Flag antibody (1/500) for 2 h and Cy3-labeled secondary antibody (1/300) for 1 h. Images were captured with the Olympus confocal microscopy.
Transient transfection, viral infection, and determination of antiviral activity
MDCK cells were seeded onto six-well plates and transfected with pSH3 or PNF empty vector at 60% confluence as previously described [26]. After 48 h of transfection, MDCK cells were infected with influenza A viruses at a multiplicity of infection (MOI) of 0.001 in serum-free DMEM containing 1 ug/ml TPCK-trypsin (Worthington, Freehold, NJ, USA) and incubated at 37°C. An aliquot of the supernatant was harvested every 12 h and virus yield was titrated by plaque assay in MDCK cells.
Western blot-based phosphorylation assay
MDCK cells transfected with indicated plasmids for 24 h were infected with different influenza A virus strains at an MOI of 2. At 8 h post infection the cells were lysed with Laemmli sample buffer containing 5 mM of NaF for 5 min in boiling water followed by brief sonication. Protein concentration was determined using an RC-DC kit (Bio-Rad, Hercules, CA, USA). Proteins (20 ug/lane) were separated in 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) with 10% non-fat milk for 2 h and incubated overnight at 4°C with anti-phospho-Akt (Ser473) (1/500) or anti-Akt (1/700) antibody. The membrane was rinsed extensively in TBST and incubated for 2 h with peroxidase-conjugated secondary antibody (1/1000). Immunoblots were developed using the Enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL, USA). Quantity One (Bio-Rad) software was used to analyze images.
Determination of PI3K effect on the replication of ST364 and PR8 viruses
MDCK cells with 100% confluence were pretreated with 10 uM or 50 uM LY294002 (a specific PI3K inhibitor). After 2 h, MDCK cells were infected with ST364 virus or PR8 virus, and plaque assay was performed.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Conceived and designed the experiments: KSL, DGZ, WZL, GFW. Performed the experiments: DGZ, WZL, GFW, CZ, XXC, YXX. Analyzed the data: DGZ, WZL, KSL, GFW, YS, XXZ. Wrote the paper: DGZ, WZL, KSL. All authors read and approved the final manuscript.
Acknowledgements
We thank Dr. William Ba-Thein for critical discussion and manuscript editing. This study was supported by National Natural Science Foundation of China (30771988, 30972766), Guangdong Natural Science Foundation(8151503102000022, 9451503102003499), Specialized Research Fund for the Doctoral Program of Higher Education (20094402110004), Outstanding Young Scientists Foundation of Guangdong Province Education Department (LYM08056), State Key Lab of Agriculture Microbiology Open Foundation (AML200910), Shantou University Medical College Research Foundation, and 211 Project of Guangdong Province (Mechanism and Prevention of Emerging Infectious Diseases).
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