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Biomedical subjects

William Marshall

Publications and source records attributed to William Marshall.

11 recordsLinked to original sources

Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

Base Sequence↗

High finesse opto-mechanical cavity with a movable thirty-micron-size mirror.

We report on the demonstration of a high finesse micro-optomechanical system and identify potential applications ranging from optical cooling to weak force detection to massive quantum superpositions. The system consists of a high quality diameter flat dielectric mirror cut from a larger substrate with a focused ion beam and attached to an atomic force microscope cantilever. Cavity ring-down measurements performed on a 25 mm long Fabry-Pérot cavity with the 30 microm mirror at one end show an optical finesse of 2100. Numerical calculations show that the finesse is not diffraction limited and that orders of magnitude higher finesse should be possible. A mechanical quality factor of more than 10(5) at pressures below 10(-3) mbar is demonstrated for the cantilever with a mirror attached.

Journal Article↗

The vaccinia virus N1L protein influences cytokine secretion in vitro after infection.

The vaccinia virus N1L ORF encodes a protein that enhances virulence and replication of the virus by an unknown mechanism. It has been studied for its ability to enhance viral replication and dissemination in the brain and more recently has been linked to an immunomodulatory role in which it inhibits the activation of cytokine transcription activators in Toll-like receptor signaling pathways after pathogen recognition. The effect of N1L on the release of cytokines from human primary monocytes was investigated. Secretion of the proinflammatory, antiviral cytokines TNF-alpha, IL-1beta, IFN-alpha, IFN-beta, and the anti-inflammatory cytokine IL-10 was found to be inhibited by the presence of the N1L protein.

Animals↗

Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference.

To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.

Clathrin↗

Towards quantum superpositions of a mirror.

We propose an experiment for creating quantum superposition states involving of the order of 10(14) atoms via the interaction of a single photon with a tiny mirror. This mirror, mounted on a high-quality mechanical oscillator, is part of a high-finesse optical cavity which forms one arm of a Michelson interferometer. By observing the interference of the photon only, one can study the creation and decoherence of superpositions involving the mirror. A detailed analysis of the requirements shows that the experiment is within reach using a combination of state-of-the-art technologies.

Journal Article↗

Can child deaths be prevented? The Arizona Child Fatality Review Program experience.

OBJECTIVE: To determine the causes and preventability of child deaths; to assess the accuracy of death certificate information; and to assess the number of child abuse deaths that are misdiagnosed as deaths attributable to natural or accidental causes. METHODS: Analysis of deaths of children <18 years old that occurred between 1995-1999 using the data collected by the Arizona Child Fatality Review Program (ACFRP). RESULTS: From 1995-1999, local multidisciplinary child fatality review teams (CFRTs) have reviewed 95% of all deaths of children <18 years old in Arizona. Each team has access to the child's death certificate, autopsy report, hospital records, child protective services records, law enforcement reports, and any other relevant documents that provide insight into the cause and preventability of a child's death. After reviewing these documents, the team determines the cause of death, its preventability, and the accuracy of the death certificate. The ACFRP defines a child's death as preventable if an individual or the community could reasonably have done something that would have changed the circumstances that led to the child's death. The ACFRP determined that 29% (1416/4806) of these deaths could have been prevented, and preventability increased with the age of the child. Only 5% (81/1781) of neonatal deaths were considered preventable, whereas the deaths of 38% of all children older than 28 days were considered preventable. By 9 years of age, the majority of child deaths (56%) were considered preventable. Deaths attributable to medical conditions were far less likely to be considered preventable than deaths attributable to unintentional injuries. Although 62% of all deaths in Arizona during the 5-year period were attributable to medical conditions, only 8% (253/2983) of these deaths were considered preventable. In contrast, 91% (852/934) of the deaths attributable to unintentional injuries were considered preventable. Motor vehicle crashes accounted for 634 of the deaths resulting from injuries, and drowning accounted for 187 deaths. Motor vehicle crashes were the leading cause of death for all children in Arizona over 1 year of age. Only 18% of child passengers and 3% of adolescent drivers who died were known to be appropriately restrained. The typical drowning victim was a young child who drowned in the family's backyard pool. Indeed, 70% (131/187) of the drowning victims were <5 years old, and 62% (81/131) of these children died in a backyard pool. Supervision of the child and pool fencing could have prevented 90% of these deaths. Most deaths attributable to medical conditions occurred in the first year of life. Prematurity was the most common medical condition (1036 deaths) followed by congenital anomalies (662 deaths) and infectious diseases (470 deaths). Some of the reasons why CFRTs believed a medical death was preventable included inadequate emergency medical services, poor continuity of care, and delay in seeking care because of lack of health insurance. There were 4 deaths resulting from infections that were vaccine-preventable. There were 263 deaths attributable to sudden infant death syndrome. Only 38 of these infants were found lying on their back; 35 were found lying on their side. The death rate from sudden infant death syndrome decreased from 1.1 per 1000 infants <1 year of age in 1995 to 0.5 in 1999. There were 33 deaths that the CFRTs concluded were attributable to unsafe sleeping arrangements that resulted in unintentional suffocation. From 1995-1999, 317 Arizona children died from gun shot wounds. Most of these deaths were homicides (175) or suicides (109). All suicide deaths occurred in children >9 years old, and 77% of these children were >14 years old. The typical suicide victim was male (83%) and used a gun (70%) to kill himself. After review by the CFRTs, it was determined that 5 of the 67 child abuse deaths were misdiagnosed as attributable to natural or accidental causes on the death certificate. In 3 of these 5 cases, the child was in a persistent vegetative state and died many years after the episode of child abuse. Although inaction or inappropriate action by Child Protective Services (CPS) is often thought to be the cause of child abuse deaths, the ACFRP determined that in 79% of child abuse deaths, there had been no previous CPS involvement with the child's family. Although 61% of child abuse deaths were considered to be preventable, much of the responsibility for preventing these deaths rests with community members (eg, relatives, neighbors) who were aware of the abuse but failed to report the family to CPS. The CFRTs, who had received training in the proper completion of death certificates, reported that the cause of death was incorrect on 13% of all death certificates and in 16 cases, the CFRTs disagreed with the medical examiner on the manner of death (eg, natural, accidental, undetermined). Because CFRTs have access to additional information that may not have been available to the physician who completes a child's death certificate, CFRTs may be able to more accurately determine the cause and manner of death than the physician who completed the death certificate. CONCLUSIONS: Arizona's child death rate is above the national average (82.16/100 000), but the ACFRP determined that many of these deaths could have been prevented by using known prevention strategies (eg, child safety restraints, pool fencing). Most child mortality data are based on death certificate information that often is incorrect and cannot be used to assess preventability. Although most states have child fatality review programs that review suspected child abuse deaths, <3% of all preventable deaths in Arizona were attributable to child abuse. If all child deaths in the United States were reviewed from a prevention/needs assessment perspective, targeted and data-driven recommendations for prevention could be developed for each community, and potentially 38% of all child deaths that occur after the first month of life could be prevented. The ACFRP is an excellent example of a statewide system with a public health focus. To assist other states in developing similar programs, national support is needed. The establishment of a public health focused federally funded national program would provide us with the opportunity to standardize data collection among states and better utilize this data at a national level.

Accidents, Traffic↗