The potential for tree planting strategies to reduce local and regional ecosystem impacts of agricultural ammonia emissions.

Trees are very effective at capturing both gaseous and particulate pollutants from the atmosphere. But while studies have often focussed on PM and NOx in the urban environment, little research has been carried out on the tree effect of capturing gaseous emissions of ammonia in the rural landscape. To examine the removal or scavenging of ammonia by trees a long-range atmospheric model (FRAME) was used to compare two strategies that could be used in emission reduction policies anywhere in the world where nitrogen pollution from agriculture is a problem. One strategy was to reduce the emission source strength of livestock management systems by implementing two 'tree-capture' systems scenarios - tree belts downwind of housing and managing livestock under trees. This emission reduction can be described as an 'on-farm' emission reduction policy, as ammonia is 'stopped' from dispersion outside the farm boundaries. The second strategy was to apply an afforestation policy targeting areas of high ammonia emission through two planting scenarios of increasing afforestation by 25% and 50%. Both strategies use trees with the aim of intercepting NH3 emissions to protect semi-natural areas. Scenarios for on-farm emission reductions showed national reductions in nitrogen deposition to semi-natural areas of 0.14% (0.2 kt N-NHx) to 2.2% (3.15 kt N-NHx). Scenarios mitigating emissions from cattle and pig housing gave the highest reductions. The afforestation strategy showed national reductions of 6% (8.4 kt N-NHx) to 11% (15.7 kt N-NHx) for 25% and 50% afforestation scenarios respectively. Increased capture by the planted trees also showed an added benefit of reducing long range effects including a decrease in wet deposition up to 3.7 kt N-NHx (4.6%) and a decrease in export from the UK up to 8.3 kt N-NHx (6.8%).

Inhibition of the IKK/NF-κB pathway by AAV gene transfer improves muscle regeneration in older mdx mice.

The IκB kinase (IKKα, ß and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKß (IKKß-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKß-dn, but not by IKK wild-type controls (IKKα and ß) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKß-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKß-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKß-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKß in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.

Improvement of the mdx mouse dystrophic phenotype by systemic in utero AAV8 delivery of a minidystrophin gene.

Duchenne muscular dystrophy (DMD) is a devastating primary muscle disease with pathological changes in skeletal muscle that are ongoing at the time of birth. Progressive deterioration in striated muscle function in affected individuals ultimately results in early death due to cardio-pulmonary failure. As affected individuals can be identified before birth by prenatal genetic testing for DMD, gene replacement treatment can be started in utero. This approach offers the possibility of preventing pathological changes in muscle that begin early in life. To test in utero gene transfer in the mdx mouse model of DMD, a minidystrophin gene driven by the human cytomegalovirus promoter was delivered systemically by an intraperitoneal injection to the fetus at embryonic day 16. Treated mdx mice studied at 9 weeks after birth showed widespread expression of recombinant dystrophin in skeletal muscle, restoration of the dystrophin-associated glycoprotein complex in dystrophin-expressing muscle fibers, improved muscle pathology, and functional benefit to the transduced diaphragm compared with untreated littermate controls. These results support the potential of the AAV8 vector to efficiently cross the blood vessel barrier to achieve systemic gene transfer to skeletal muscle in utero in a mouse model of muscular dystrophy, to significantly improve the dystrophic phenotype and to ameliorate the processes that lead to exhaustion of the skeletal muscle regenerative capacity.

Full-length dystrophin gene transfer to the mdx mouse in utero.

In utero gene therapy for genetic diseases, such as muscular dystrophies, offers potential advantages over postnatal treatment including vector delivery at the earliest point in the disease and treatment prior to full maturation of the immune system. This study examines in utero gene delivery of full-length murine dystrophin to the murine mdx model for Duchenne muscular dystrophy using a high-capacity adenoviral vector. We examined dystrophin expression, spread of vector, morphology and specific force production of the tibialis anterior muscle 9 weeks after intramuscular in utero injection. Recombinant dystrophin was expressed in the hindlimb muscles, with the majority of animals having expression in two muscles of the injected hindlimb. The dystrophin-glycoprotein complex was restored in those muscle fibers expressing recombinant dystrophin. Analysis of the percentage of dystrophin-expressing muscle fibers with centrally placed nuclei revealed effective protection from cycles of degeneration and regeneration normally seen in muscle fibers lacking dystrophin. However, due to low levels of muscle gene transfer, further advances in the efficiency of adenoviral vector-mediated gene delivery would be required for clinical applications of in utero gene therapy for primary myopathies such as Duchenne muscular dystrophy.

Comparison of high-capacity and first-generation adenoviral vector gene delivery to murine muscle in utero.

In utero gene delivery could offer the advantage of treatment at an early stage for genetic disorders such as Duchenne muscular dystrophy (DMD) in which the inevitable process of muscle degeneration is already initiated at birth. Furthermore, treatment of fetal muscle with adenoviral (Ad) vectors is attractive because of a high density of Ad receptors, easy vector accessibility due to immaturity of the basal lamina and the possibility of treating stem cells. Previously, we demonstrated the efficient transduction of fetal muscle by high-capacity Ad (HC-Ad) vectors. In this study, we compared HC-Ad and first-generation Ad (FG-Ad) vectors for longevity of lacZ transgene expression, toxicity and induction of immunity after direct vector-mediated in utero gene delivery to fetal C57BL/6 mice muscle 16 days after conception (E-16). The total amount of beta-galactosidase (betagal) expressed from the HC-Ad vector remained stable for the 5 months of the study, although the concentration of betagal decreased due to muscle growth. Higher survival rates that reflect lower levels of toxicity were observed in those mice transduced with an HC-Ad vector as compared to an FG-Ad vector. The toxicity induced by FG-Ad vector gene delivery was dependent on mouse strain and vector dose. Animals treated with either HC-Ad and FG-Ad vectors developed non-neutralizing antibodies against Ad capsid and antibodies against betagal, but these antibodies did not cause loss of vector genomes from transduced muscle. In a mouse model of DMD, dystrophin gene transfer to muscle in utero using an HC-Ad vector restored the dystrophin-associated glycoproteins. Our results demonstrate that long-term transgene expression can be achieved by HC-Ad vector-mediated gene delivery to fetal muscle, although strategies of vector integration may need to be considered to accommodate muscle growth.

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Local high-capacity adenovirus-mediated mCTLA4Ig and mCD40Ig expression prolongs recombinant gene expression in skeletal muscle.

Multiple forms of muscular dystrophy are due to the absence of cytoskeletal muscle proteins that normally protect the integrity of muscle cells. The lack of any adequate treatments for these devastating diseases propels research toward the development of strategies for gene delivery to skeletal muscle. High-capacity adenoviral vectors (HC-AdV) devoid of all viral coding sequences have been developed to avoid expression of viral proteins by the gene therapy vector. However, the capsid proteins that are an essential component of the input viral vector and any residual helper virus in the vector preparation could induce an immune response. Furthermore, the therapeutic protein provided by a gene transfer vector presents the potential to induce an immune response in a patient who does not express a normal cellular protein due to genetic mutation. Therefore, we hypothesize that some immune suppression will be required with therapeutic gene delivery designed for the treatment of patients with inherited muscle diseases. In this study, we constructed and rescued three HC-AdVs expressing murine CTLA4Ig, murine CD40Ig, or both. The backbone vector without a gene insert was rescued as a negative control vector. The production of relevant proteins from each vector was determined in vitro. In vivo function of each of the immunosuppressant vectors was assayed by co-injection with an enhanced green fluorescent protein (EGFP)-expressing first-generation adenoviral vector (AdEGFP) into the tibialis anterior muscle of C57BL/10 mice. Higher levels of muscle EGFP expression were observed in animals receiving an immunosuppressant vector. Furthermore, the production of total anti-AdV and anti-EGFP antibodies was reduced in mice treated with each of the three immunosuppressant vectors. A second intramuscular administration of AdEGFP alone 4 weeks after the initial co-injection was successful in all immunosuppressant vector-treated groups, but not in the negative control vector-treated group. All groups had a high antibody response to adenoviral proteins after the second injection of AdEGFP alone, indicating that the initial co-injection did not tolerize against vector capsid antigens.

The role of autopsy on patients with burns.

Burn center verification requires the use of autopsy as one method of quality assurance in a burn center. Because of the decreasing rates of autopsies worldwide and improved diagnostic accuracy in our critical care units, we tested the hypothesis that autopsy diagnosis would not alter our clinical diagnosis. A chart review of all deaths (N = 94) that occurred during a 6-year period (1989-1994) was performed. The clinical diagnoses from the hospital charts and autopsy reports for the patients were reviewed, and diagnostic discrepancies were classified as class I or class II errors. Class I diagnostic errors might have altered the clinical outcome. Class II errors were attributable to the burn injuries but were believed to have had little impact on the clinical outcome. The overall autopsy rate was 93.6% (n = 88). Clinical diagnostic errors were found in 16 (18%) of 88 patients. Five class I errors were found in 4 patients (4.5%), and 15 class II errors were found in 13 patients (14.7%). Although the rate of potentially serious errors was low (only 4.5% of the patients in this study) postmortem examinations revealed clinical diagnostic errors. The results of this study support the continued use of autopsies as a means of quality assurance, despite our ability to closely monitor our critically ill patients with burns.

Self-inflicted and unintentional firearm injuries among children and adolescents: the source of the firearm.

CONTEXT: The source and ownership of guns used by children to shoot themselves or others is largely unknown. OBJECTIVE: To determine the ownership and usual storage location of firearms used in unintentional and self-inflicted intentional firearm deaths and injuries. DESIGN: Retrospective case series. SETTING: King County, Washington. PATIENTS: Youths aged from birth to 19 years who sought medical treatment at a level I trauma center for a self-inflicted or unintentional firearm injury between 1990 and 1995 or who presented to the county medical examiner with a fatal self-inflicted or unintentional firearm injury between 1990 and 1995. DATA SOURCES: County medical examiner records, regional police investigative reports, medical records from a level I trauma center, and surveys of victims' families. MAIN OUTCOME MEASURES: Source and ownership of the associated firearm. RESULTS: Fifty-six fatal injuries and 68 nonfatal firearm injuries that met the criteria were identified. Of these, 59 were intentionally self-inflicted deaths and injuries and 65 were unintentional deaths and injuries. A firearm owned by a household member living with the victim was used in 33 (65%) of 51 suicides and suicide attempts and 11 (23%) of 47 unintentional injuries and deaths. Additionally, a firearm owned by another relative, friend, or parent of a friend of the victim was used in 4 (8%) of the 51 suicides and suicide attempts and 23 (49%) of the 47 unintentional injuries and deaths. Parental ownership accounted for 29 (57%) of the 51 suicides and suicide attempts and 9 (19%) of the 47 unintentional injuries and deaths. More than 75% of the guns used in suicide attempts and unintentional injuries were stored in the residence of the victim, a relative, or a friend. CONCLUSION: Most guns involved in self-inflicted and unintentional firearm injuries originate either from the victim's home or the home of a friend or relative.

Temperature dependence of inorganic nitrogen uptake: reduced affinity for nitrate at suboptimal temperatures in both algae and bacteria.

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 approximately 3, where Q10 is the proportional change with a 10 degrees C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures.

Artifactual injuries of the larynx produced by resuscitative intubation.

Over a period of 9 months we examined a series of 50 deaths due to natural and unnatural causes in which there had been endotracheal intubation and chest compression during resuscitation at the scene or in the emergency department shortly before death. In 37 of 50 cases (74%) there were airway injuries directly resulting from the intubation procedure which we documented using a standardized protocol and photography. Specific airway injuries, ranging from petechiae to contusions, included oral injury (28%), posterior pharyngeal injury (16%), epiglottic injury (22%), piriform recess injury (12%), laryngeal and tracheal mucosa injury (64%), strap muscle hemorrhage (14%), and cutaneous injury of the neck (4%). In addition, we recorded the presence of facial (6%) and conjunctival petechiae (21%) and attributed these changes to resuscitative chest compression. No cases had associated fractures of the hyoid or thyroid cartilage. Based on our findings, we conclude that resuscitative intubation can cause artifactual injury that may mimic inflicted injuries caused by neck compression, including strangulation and neck holds.

Comments on "A new approach to adaptive fuzzy control: the controller output error method".

For original paper see ibid., vol 27, p. 686-91, Aug. 1997. In the above paper, a novel algorithm for adaptively updating the parameters of a fuzzy controller was proposed. The purpose of this letter is to point out that this algorithm, and its use, are well known. The authors of the above paper acknowledge the previous use of similar concepts, however this letter draws attention to a particularly clear description of the algorithm.

Death in custody.

Death in custody is the most demanding investigation that a medical examiner or coroner can perform. The investigation requires attention to detail at the time of scene investigation and autopsy examination, as well as a careful assessment of the toxicologic results and circumstances of the death. Synthesis of the many facts that are developed during the investigation should allow the medical examiner or coroner to establish a reasonable cause of death. There can be legitimate points of disagreement of interpretation because the conclusions so frequently are predicated on physiologic processes and not on anatomic findings. It is incumbent on the medical examiner and coroner responsible for investigating deaths of these types to utilize all the information generated during the investigation and to identify an appropriate cause of death.

Accidental firearm fatality involving a hand-crafted pen gun. Case report.

We report a case in which a homemade 0.25 caliber "pen gun" resulted in the accidental death of a man examining the weapon unaware of its lethal nature. The design of the weapon consisted of a spring-loaded firing mechanism contained in a housing resembling a writing pen. Thus, the weapon was similar to zip guns and tear gas pen guns but differed in its craftsmanship and primary purpose of firing conventional ammunition. Another notable feature of the case was an atypical entrance wound due to the 0.25 caliber Winchester AXP ammunition that the pen gun fired. With respect to the weapon and ammunition, this case represents an unusual firearm fatality reported to increase awareness and alert pathologists, as well as law enforcement personnel, to the existence of homemade weapons made to look like innocuous objects.

Postmortem perianal findings in children.

The postmortem finding of anal dilation or an exposed pectinate line in children who have died under suspicious circumstances continues to raise the concern of possible sexual abuse. The following multicenter, collaborative study was designed to help address that question. Sixty-five subjects, ranging in age from birth to 17 years, were autopsied at three different sites. A standard protocol along with 35-mm cameras were used to record the results. Thirty-eight (58%) subjects were boys, and 27 (42%) were girls. Forty-two (65%) were white, 10 (15%) African-American, five (8%) Asian, three (5%) white Hispanic and five (8%) other. Fifty-seven (88%) were in Tanner stage I of secondary sexual development. Thirty-four (52%) died of natural causes, 26 (40%) from accidental injuries, three (5%) from other causes, and four (6%) as a result of a homicide. Forty-eight subjects (74%) had some dilation of the anal sphincters. In 21 children (32%), the entire anal canal, including the rectal ampulla, could be visualized. In another 21 (32%) subjects, the pectinate line was exposed. Only the outer portion of the anal canal opened in six children (10%), whereas 17 (26%) had no dilatation of the anus. Anal laxity led to flattened skin folds in 50 (77%), a shallow anal canal in 40 (62%), the exposure of both the pectinate line in 38 (59%), and the anal mucosa in 24 (37%). Venous congestion was present in 14 (22%), venous pooling in three (5%), erythema in six (9%), and increased pigmentation in eight (12%). Funneling was found in two (3%). Blood was present in three (5%), and an abrasion was discovered in one (2%). No fissures, lacerations, hemorrhoids, or scars were found in any of the children. Anal orifice size varied with the age of the child, the amount of traction applied to the buttocks, and a history of a CNS injury at the time of death. It is suggested, finally, that anal dilatation alone cannot be used a marker for prior sexual abuse and the exposure of the pectinate line should not be confused with tears or fissures of the anal verge. Further studies of children known to have been sodomized prior to death are required.

Asphyxial suicides using plastic bags.

Fifty-three suicides using plastic bags were identified in a review of cases within the jurisdiction of the King County Medical Examiner's Office, Seattle, Washington from 1984 to 1993. We found that this method was used at a greater frequency by individuals older than 50 in comparison with other methods. The most commonly identified stressor leading to the suicide in this population was failing health. The use of this method as a means of "self deliverance," as advocated by the Hemlock Society, could be inferred in only a small minority of cases where terminal illnesses were identified. This method may be preferred by those older than 50 years because of the ready availability of plastic bags and the relative nonviolence of the death. Analysis of the autopsy findings showed no specific features for this method of suicide. In particular, petechiae, which are often considered a marker of asphyxia, were present in only a small minority of cases (3%). Furthermore, the scene investigation rarely revealed specific features, other than the plastic bag in place. Thus, if the plastic bag were removed after death, the cause and manner of death would be obscure.

Prone sleep position and the sudden infant death syndrome in King County, Washington: a case-control study.

OBJECTIVE: To determine whether the prone sleep position was associated with an increased risk of the sudden infant death syndrome (SIDS). STUDY DESIGN: Population-based case-control study. PARTICIPANTS: Case subjects were infants who died of SIDS in King County, Washington. Control subjects were randomly selected infants born in King County. Up to four control subjects were matched on date of birth to each case subject. METHODS: During the study period, November 1992 through October 1994, sleep-position data were collected on infants who died of SIDS by the King Count Medical Examiner's Office during their investigation of the deaths. Parents of infants chosen as control subjects were contacted by telephone, and sleep position information was obtained. Infants who usually slept on their abdomen were classified as sleeping prone; those who usually slept on the side or back were categorized as sleeping nonprone. The adjusted odds ratio for prone sleep position as a risk factor for SIDS was calculated with conditional logistic regression after control for race, birth weight, maternal age, maternal marital status, household income, and maternal cigarette smoking during pregnancy. RESULTS: Sleep position data were collected on 47 infants with SIDS (77% of eligible infants) and 142 matched control subjects; 57.4% of infants who died of SIDS usually slept prone versus 24.6% of control subjects (p < 0.00001). The unadjusted odds ratio for prone sleep position as a risk factor for SIDS was 4.69 (95% confidence interval: 2.17, 10.17). After control for potentially confounding variables, the adjusted odds ratio for prone sleep position was 3.12 (95% confidence interval: 1.08, 9.03). CONCLUSION: Prone sleep position was significantly associated with an increased risk of SIDS among a group of American infants.