Machine-guided design of synthetic cell type-specific cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing, and stimulus responses, which collectively define the thousands of unique cell types in the body. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for an intended purpose has arisen naturally through evolution. Here, we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell type specificity. We leverage innovations in deep neural network modeling of CRE activity across three cell types, efficient in silico optimization, and massively parallel reporter assays (MPRAs) to design and empirically test thousands of CREs. Through in vitro and in vivo validation, we show that synthetic sequences outperform natural sequences from the human genome in driving cell type-specific expression. Synthetic sequences leverage unique sequence syntax to promote activity in the on-target cell type and simultaneously reduce activity in off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs and demonstrate the required literacy to write regulatory code that is fit-for-purpose in vivo across vertebrates.

Survival, Differentiation, and Migration of High-Purity Mouse Embryonic Stem Cell-derived Progenitor Motor Neurons in Fibrin Scaffolds after Sub-Acute Spinal Cord Injury.

Embryonic stem (ES) cells can be differentiated into many neural cell types that hold great potential as cell replacement therapies following spinal cord injury (SCI). Coupling stem cell transplantation with biomaterial scaffolds can produce a unified combination therapy with several potential advantages including enhanced cell survival, greater transplant retention, reduced scarring, and improved integration at the transplant/host interface. Undesired cell types, however, are commonly present in ES-cell derived cultures due to the limited efficiency of most ES cell induction protocols. Heterogeneous cell populations can confound the interaction between the biomaterial and specific neural populations leading to undesired outcomes. In particular, biomaterials scaffolds may enhance tumor formation by promoting survival and proliferation of undifferentiated ES cells that can persist after induction. Methods for purification of specific ES cell-derived neural populations are necessary to recognize the full potential of combination therapies involving biomaterials and ES cell-derived neural populations. We previously developed a method for enriching ES cell-derived progenitor motor neurons (pMNs) induced from mouse ES cells via antibiotic selection and showed that the enriched cell populations are depleted of pluripotent stem cells. In this study, we demonstrate the survival and differentiation of enriched pMNs within three dimensional (3D) fibrin scaffolds in vitro and when transplanted into a sub-acute dorsal hemisection model of SCI into neurons, oligodendrocytes and astrocytes.

Suicide among North Carolina women, 1989-93: information from two data sources.

OBJECTIVES: To characterize the events and examine suicide precursors among women and to examine gaps in surveillance. SETTING: A statewide study in North Carolina. METHODS: Suicides of women age 15 and older for the time period 1989-93, as identified from the Office of the Chief Medical Examiner, were included. All case files were reviewed by hand and telephone interviews were attempted with investigating law enforcement officials for every case in 1993. RESULTS: Altogether 882 suicides met the case definition, for an age adjusted rate that fluctuated between 5.53 and 7.26 per 100 000 women across the period. Interviews with law enforcement officials were completed for 135 of the 177 cases from 1993. White women had rates nearly three times those of racial minorities. Women under age 45 were proportionally more likely than older women to have recently experienced the breakup of an intimate relationship. Information about precursors was not as consistently reported as had been hoped. Medical examiner records were variable in completeness. Law enforcement interviews frequently did not yield information about the factors we had hoped to examine, probably because the investigations were conducted primarily to rule out homicide. CONCLUSIONS: This study suggests somewhat different precursor patterns by age group. It also points to the need for reconsidering how suicide surveillance is accomplished as a strategy to guide intervention.

Comparison of commercially available three-dimensional treatment planning algorithms for monitor unit calculations in the presence of heterogeneities.

This study uses an anthropomorphic phantom and its computed tomography (CT) data set to evaluate monitor unit (MU) calculations using the CMS Focus Clarkson, the CMS Focus Multigrid Superposition Model, the CMS Focus FFT Convolution Model, and the ADAC Pinnacle Collapsed Cone Convolution Superposition Algorithms. Using heterogeneity corrections, a treatment plan and corresponding MU calculations were generated for several typical clinical situations. A diode detector, placed in an anthropomorphic phantom, was used to compare the treatment planning algorithms' predicted doses with measured data. Differences between diode measurements and the algorithms' calculations were within reasonable levels of acceptability as recommended by Van Dyk et al. [Int. J. Rad. Onc. Biol. Phys. 26, 261-273 (1993)], except for the CMS Clarkson algorithm, which predicted too few MU for delivery of the intended dose to chest wall fields.

In vitro susceptibilities of Bartonella and Rickettsia spp. to fluoroquinolone antibiotics as determined by immunofluorescent antibody analysis of infected Vero cell monolayers.

The in vitro susceptibilities of Bartonella and Rickettsia spp. to different concentrations of ciprofloxacin, levofloxacin, ofloxacin and sparfloxacin in Vero cell cultures, were determined by enumeration of immunofluorescent-stained bacilli. After incubation in a CO(2)-enriched atmosphere, inocula were replaced and tested with media containing 12 different concentrations of each antibiotic in replicate for each species and the monolayers were re-incubated. Growth status was determined by evaluation of immunofluorescent staining bacilli. Effective inhibitory antibiotic dilution endpoints were determined by counting Bartonella- and Rickettsia-specific fluorescent foci across a range of antibiotic dilutions with an epi-fluorescent microscope, and were compared with an antibiotic-negative control. Based upon the use of C(max):MIC and AUC:MIC data, levofloxacin exhibited activity against Bartonella elizabethae and B. quintana.

Homicide on the job: workplace and community determinants.

Homicide is the second leading cause of death on the job for workers in the United States. To identify workplace-level predictors of homicide risk, a case-control study of worker killings in North Carolina in 1994-1998 was conducted. Workplaces were the units of analysis: case workplaces (n = 105) were those where a worker was killed during the study period, while controls (n = 210) were a density sample of North Carolina workplaces, matched on time and industry sector. Potential risk and protective factors were assessed in telephone interviews with workplace managers. Associations were measured by the exposure odds ratio and 95% confidence interval, estimated via conditional logistic regression. Characteristics associated with notably higher risk included being at the current location for 2 years or less (odds ratio (OR) = 5.3, 95% confidence interval (CI): 2.2, 12.6), having only one worker (OR = 2.9, 95% CI: 1.2, 7.2), and having night (OR = 4.9, 95% CI: 2.7, 8.8) or Saturday (OR = 4.2, 95% CI: 1.9, 9.2) hours. Workplaces with only male employees (OR = 3.1, 95% CI: 1.5, 6.5) or with African-American or Asian employees were also more likely to experience a killing. While few of the preceding risk factors are directly modifiable through workplace interventions, it is important to identify them before developing or evaluating preventive measures.

Outcomes of comfort touch in institutionalized elderly female residents.

Comfort touch as a holistic nursing intervention has gained considerable attention in the past decade. The purpose of this experimental study was to examine whether comfort touch improved the perceptions of self-esteem, well-being and social processes, health status, life satisfaction and self-actualization, and faith or belief and self-responsibility in 45 institutionalized elderly female residents. Results revealed that comfort touch significantly improved the perceptions of all elements. This article offers recommendations for practice, research, and education.

A population based study of unintentional firearm fatalities.

OBJECTIVES: To describe the circumstances of fatal unintentional firearm injuries in a statewide population in a region of the United States with high firearm fatality rates and to compare to similar data from an earlier period in the same state. METHODS: Analyses of North Carolina medical examiner database (1985-94) and review of medical examiner case reports (1990-94) and comparison to similar data from 1979-82. RESULTS: A total of 390 unintentional shooting deaths occurred (0.59/100,000 population) between 1985-94 with the highest rate in the ages 15-24. Between 1990-94, handguns were responsible for 59% of these deaths compared to 40% in the 1979-82 period. Younger victims were more likely to be shot by family or friends, though, 53% of all deaths were self inflicted. In 45 cases, the person firing the weapon was reported to believe that the gun was unloaded or had the safety device activated. CONCLUSIONS: This study demonstrates changes in patterns of unintentional firearm fatalities in North Carolina in two decades, particularly the increase in incidence of events involving handguns. The results highlight the need for additional attention to efforts governing access to firearms, particularly handguns; technological advances in designing safer guns, and additional emphasis on safe storage policies and practices.

Disposition and functions of clarithromycin in human THP-1 monocytes during stimulated and unstimulated conditions.

The macrolide antibiotics are bacteriostatic agents interfering with protein synthesis but they are taken up by phagocytic cells, e.g. macrophages, neutrophils and fibroblasts which take up infectious organisms into phagosome-lysosomal vaculoes. Recent studies have suggested that these macrolide antibiotics block the spread of infections by mechanisms associated with the inflammation process. Herein is a study with clarithromycin using human THP-1 monocytes, a phagocytic cell which has not been studied to date. Clarithromycin was rapidly taken up by the monocytes (approximately 1%) utilizing both saturable carrier and passive processes at pH 7.4 but was exclusively passive at pH 6.8 and 5.0. The carrier process was energy and temperature dependent and appeared to be linked to certain ion channels. Efflux of the drug was rapid and complete in 1 hr. Intracellular disposition showed 74% in the cell sap and 11% in the nucleus. Upon stimulation with zymogen A or bacteria significant increases of uptake occurred in the isolated lysosome-phagosomes. Examination showed that initially clarithromycin treatment triggered the release of NO, H2O2, IL-1 and TNFalpha from the monocytes, known mediators of inflammation, but also mediators which cause bacterial cell death or apoptosis. The activity of the monocyte marker hydrolytic enzyme NAG was elevated at this time as well as protein kinase C activity. Treatment from 2-4 hr with clarithromycin appeared to reverse this process in that the chemical mediator release was reduced along with the activities of hydrolytic enzymes, e.g. NAG and cathepsin D with no evidence of lipid peroxidation and protective SOD enzyme activity elevation. The latter effects of the antibiotic would be useful in blocking the spread of infection or inflammation from the original site. The normal bacterial static killing effects of clarithromycin was evident at 24 but not 2 hr in both extracellular free bacteria and those bacteria phagocytosed by the THP-1 monocytes.

Paclitaxel, carboplatin, and vinorelbine in the treatment of advanced non-small cell lung cancer: a phase II trial of the Minnie Pearl Cancer Research Network.

PURPOSE: To evaluate the feasibility, toxicity, and efficacy of adding vinorelbine to the paclitaxel/carboplatin combination in the treatment of advanced non-small cell lung cancer. PATIENTS AND METHODS: Patients with advanced (stage IIIB/IV) non-small cell lung cancer who had received no previous chemotherapy were treated with the following three-drug regimen: paclitaxel, 200 mg/m2, 1-hour i.v. infusion, day 1; carboplatin, AUC 6.0 i.v., day 1; and vinorelbine, 22.5 mg/m2 i.v. days 1 and either 8 or 15. Treatment was repeated every 21 days. This phase II trial was conducted in a multicenter, community-based setting. RESULTS: Eighty-nine patients were treated with a median of four courses of therapy (range, one to eight). Thirty-one patients (35%) had major responses (two complete, 29 partial), and 36 patients (40%) had a minor response or stable disease. Actuarial median survival was 8.6 months; 1 year survival was 43%. Leukopenia was the major toxicity: 73% of patients had grade 3/4 toxicity, and 32 patients (36%) were hospitalized for neutropenia/fever (11% of total courses administered). Treatment-related death due to infection occurred in four patients (4%). CONCLUSIONS: This three-drug regimen is feasible and efficacious in the treatment of advanced non-small cell lung cancer. The addition of vinorelbine increases the incidence of severe leukopenia substantially when compared with the paclitaxel/carboplatin regimen. However, other toxicities are not markedly increased. Ongoing randomized trials will define the role of this regimen in the treatment of non-small cell lung cancer.

Killed on the clock: a population-based study of workplace homicide, 1977-1991.

BACKGROUND: This paper describes the epidemiology of workplace homicides in North Carolina, with emphasis on the circumstances. METHODS: Workplace homicide victims were identified by and data were abstracted from the North Carolina medical examiner system. RESULTS: Workplace homicide rates are highest for men, older and self-employed workers, minorities and specific occupations, especially taxi drivers. Robberies, mostly in retail settings, accounted for half of the cases, while 20% were known to involve disputes, the contexts of which differed by sex. Women were most likely to be killed by estranged partners. CONCLUSIONS: Preventive strategies need to address the specific contexts in which workplace homicide occurs, such as retail and taxi robberies, and law enforcement officers interacting with suspects. A workplace response to domestic violence is also needed. Other areas for future research and intervention include environmental modifications, employee screening and training, and identifying more inclusive occupational data sources.

In vitro susceptibilities of Rickettsia and Bartonella spp. to 14-hydroxy-clarithromycin as determined by immunofluorescent antibody analysis of infected vero cell monolayers.

The in vitro susceptibilities of Rickettsia akari, Rickettsia conorii, Rickettsia prowazekii, Rickettsia rickettsii, Bartonella elizabethae, Bartonella henselae and Bartonella quintana to different concentrations of clarithromycin, 14-hydroxy-clarithromycin (the primary metabolite of clarithromycin) and tetracycline in Vero cell cultures, were determined by enumeration of immunofluorescently-stained bacilli. The extent of antibiotic-induced inhibition of foci was recorded for each dilution of antibiotic and compared with an antibiotic-negative control. Based upon MIC data, clarithromycin alone is highly active against all three Bartonella spp., R. akari and R. prowazekii, while 14-hydroxy-clarithromycin is active against R. conorii, R. prowazekii and R. rickettsii. Further testing is warranted in animal models and human clinical trials, to examine the activity of both clarithromycin and its primary metabolite and to define further the role of clarithromycin in therapy, particularly of infections caused by obligate intracellular bacteria such as Rickettsia and Bartonella spp.

Caregiver distress: what nurses in rural settings can do to help.

Caregivers in rural settings experience unmet needs. Some family caregivers in rural areas, either willingly or grudgingly, take on the role of caregiving as one of many responsibilities. A review of the literature reveals that the burden of the added responsibility results in physiological and psychological caregiver distress. The authors suggest some strategies to meet caregiver needs and outline areas where research is needed.

Discontinuation rates for protease inhibitor regimens containing ritonavir 600 mg versus ritonavir 400 mg plus saquinavir 400 mg.

OBJECTIVE: A retrospective study was performed to determine whether twice-daily ritonavir 400 mg plus twice-daily saquinavir 400 mg (ritonavir 400/saquinavir 400) was better tolerated than ritonavir 600 mg twice daily (ritonavir 600). A secondary objective was to determine whether the rate of discontinuation due to therapeutic failure differed between the two ritonavir regimens. DESIGN: The study was a retrospective chart review. Data collected included ritonavir dose; length of ritonavir therapy; reason for discontinuation; HIV-1 RNA prior to and at discontinuation of ritonavir therapy; CD4+ count; and antiretroviral therapy prior to, concomitant with, and initiated after ritonavir therapy. SETTING: Patient charts were reviewed in a university teaching hospital clinic. PATIENTS: Patients were identified through a search of the pharmacy database from December 18, 1995, to December 18, 1997. Patients were > 18 years old, but not restricted by gender or race. MAIN OUTCOME MEASURES: The main outcome measures were frequency of discontinuation of ritonavir due to intolerance or due to lack of therapeutic efficacy. RESULTS: The search identified 116 patients, including 57 patients taking ritonavir 400/saquinavir 400 and 54 patients taking ritonavir 600. Five patients on other ritonavir regimens were excluded. Significantly fewer patients receiving ritonavir 400/saquinavir 400 (14%) discontinued ritonavir due to intolerance compared with ritonavir 600 (37%; p = 0.002). Discontinuations due to therapeutic failure were not significantly different: 8.8% for ritonavir 400/saquinavir 400 and 7.4% for ritonavir 600, despite the fact that ritonavir/saquinavir therapy followed another protease inhibitor in 41 patients (73.2%) compared with 12 patients (24.5%) for ritonavir 600 (p = 0.001). CONCLUSIONS: Ritonavir 400/saquinavir 400 is better tolerated than ritonavir 600.

Underascertainment of child abuse mortality in the United States.

CONTEXT: Mortality figures in the United States are believed to underestimate the incidence of fatal child abuse. OBJECTIVES: To describe the true incidence of fatal child abuse, determine the proportion of child abuse deaths missed by the vital records system, and provide estimates of the extent of abuse homicides in young children. DESIGN AND SETTING: Retrospective descriptive study of child abuse homicides that occurred over a 10-year period in North Carolina from 1985-1994. CASES: The Medical Examiner Information System was searched for all cases of children younger than 11 years classified with International Classification of Diseases, Ninth Revision codes E960 to E969 as the underlying cause of death and homicide as the manner of death. A total of 273 cases were identified in the search and 259 cases were reviewed after exclusion of fetal deaths and deaths of children who were not residents of North Carolina. MAIN OUTCOME MEASURE: Child abuse homicide. RESULTS: Of the 259 homicides, 220 (84.9%) were due to child abuse, 22 (8.5%) were not related to abuse, and the status of 17 (6.6%) could not be determined. The rate of child abuse homicide increased from 1.5 per 100000 person-years in 1985 to 2.8 in 1994. Of all 259 child homicides, the state vital records system underrecorded the coding of those due to battering or abuse by 58.7%. Black children were killed at 3 times the rate of white children (4.3 per 100000 vs 1.3 per 100000). Males made up 65.5% (133/203) of the known probable assailants. Biological parents accounted for 63% of the perpetrators of fatal child abuse. From 1985 through 1996, 9467 homicides among US children younger than 11 years were estimated to be due to abuse rather than the 2973 reported. The ICD-9 cause of death coding underascertained abuse homicides by an estimated 61.6%. CONCLUSIONS: Using medical examiner data, we found that significant underascertainment of child abuse homicides in vital records systems persists despite greater societal attention to abuse fatalities. Improved recording of such incidences should be a priority so that prevention strategies can be appropriately targeted and outcomes monitored, especially in light of the increasing rates.

A subtype of sporadic prion disease mimicking fatal familial insomnia.

OBJECTIVE: To establish a variant of sporadic prion disease as the sporadic form of fatal familial insomnia (FFI). BACKGROUND: FFI is a recently described prion disease characterized clinically by severe sleep impairment, dysautonomia, and motor signs, and pathologically by atrophy of thalamic nuclei, especially the medial dorsal and anterior ventral, and of the inferior olive. FFI is linked to the D178N mutation coupled with the methionine codon at position 129 in the prion protein gene (PRNP). It is also identified by the properties of the abnormal prion protein (PrP(Sc)), which has the relative molecular mass of 19 kDa, corresponding to the so-called type 2, and a marked underrepresentation of the unglycosylated form relative to the diglycosylated and monoglycosylated forms. METHODS: Clinical, pathologic, PrP(Sc), and PRNP data from 5 subjects with a sporadic prion disease phenotypically similar to FFI were collected and analyzed. RESULTS: All 5 subjects had a disease clinically similar and histopathologically virtually identical to FFI. PrP(Sc) type 2 was present in all subjects in amount and distribution similar to those of FFI. However, the PrP(Sc) did not show the striking underrepresentation of the unglycosylated isoform of the protein that is characteristic of FFI. Moreover, none of the subjects had the D178N PRNP mutation but all were homozygous for methionine at codon 129. CONCLUSION: This condition is likely to represent the sporadic form of FFI and the term "sporadic fatal insomnia" is proposed.

Automated telephone-administered substance abuse screening for adults in primary care.

Despite a general awareness that screening for substance abuse is an important component of primary care, many clinicians are reluctant to screen for such issues. This study examines the utility of an interactive telephone technology for screening for substance abuse in primary care settings. The study was conducted at two primary care medical practices in Cleveland, Ohio. The project was implemented in two phases. The first phase examines the general reaction to the new screening methodology of 305 patients and 40 practitioners in two general medical practices. The second phase examines the concordance of patient responses given to the telecomputer screening system as compared to responses given to a research nurse in the practice. Results indicate that both patients and practitioners have generally positive reactions to this mode of screening and results may be comparable to clinician-administered screening.

Partner homicide-suicide involving female homicide victims: a population-based study in North Carolina, 1988-1992.

Homicide-suicide is a form of fatal violence in which an individual commits homicide and subsequently kills him- or herself. One hundred and sixteen homicide-suicide events involving 119 female homicide victims in North Carolina from 1988-1992 were identified through state medical examiner files. Case files were reviewed retrospectively to identify event characteristics, precursors, and typologies. In 86% of cases the perpetrator was the current or former partner of the victim. During the study period, 24% of men who killed their female partners in North Carolina subsequently committed suicide and another 3% attempted suicide but survived. Victim separation from the perpetrator was the most prevalent precursor (41%), followed by a history of domestic violence (29%). In nearly half of the cases with a history of domestic violence, the victim had previously sought protection from the perpetrator in the form of an arrest warrant, restraining order, or intervention by a law enforcement officer. Children of the victim (and/or perpetrator) witnessed the homicide-suicide, were in the immediate vicinity, found their parents' bodies, or were killed, in 43% of cases. The prevalence of separation and domestic violence suggests several potential points of intervention, including stronger domestic violence legislation. Future research should place priority on assessing the impact of partner homicide-suicides on the families in which they occur. Such studies are essential for the informed development of preventive and therapeutic interventions for the families of both the victims and perpetrators of these fatal events. In addition, research focused on assisting men in coping with issues of control and separation is needed.

Tenure: viable or a dinosaur?

This article discusses the history of tenure in academia as well as the current pros and cons of the tenure system. A review of nursing and non-nursing literature reveals that the most important goal confronting the nation's universities and colleges today is to find more creative ways to define scholarly work and incorporate it into promotion and tenure guidelines. The author presents arguments for and against tenure, reviews the impact of tenure in nursing, and offers recommendations and solutions to the tenure dilemma in nursing.