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A cursory review of relevant research works published in the Journal of Nursing Education suggests our community relies heavily on Cohen's d . Although Cohen's d is a valuable effect size, it possesses a number of limitations that warrants our community adopting a wider set of effect size statistics to help promote a valid science of nursing education. We specifically call attention to Hedges' g. [ J Nurs Educ . 2023;62(5):316–317.]

The purpose of this study was to assess the impact of coronavirus disease 2019 on perceived stress experienced by nurse practitioners/nurses and their desire to remain employed as health care providers. A cross-sectional survey of 40 questions was administered between May and September 2020 to a convenience sample of 433 nurse practitioners and nurses in Missouri and Georgia through a Qualtrics (Provo, UT) link provided via their professional organization listserv. Anxiety-related symptoms and perceived helplessness were correlated with personal protective equipment concerns and management approachability. Problematic stress was associated with willingness to leave their current job or the nursing profession altogether.

Over the course of 2022, we have encouraged our community to adopt increasingly effective methods of mitigating the harm missing data poses to a valid science of nursing education. With our last column entry for the year, we conclude our emphasis by looking at a few approaches to the prevention of problematic missingness that can be implemented prior to collecting data. [ J Nurs Educ . 2022;61(11):657–658.]

In 2023, the Methodology Corner column has opted to promote the efficacious use of effect sizes among our community of researchers. In this first entry of the year, we call attention to broad improvement in effect size reporting germane to our community, including more consistent reporting of effect sizes and their interpretations. [ J Nurs Educ . 2023;62(2):116.]

Nurse researchers have long been encouraged to consult a priori power analyses to help design studies that are robust to type II errors. Unfortunately, a cursory look at Journal of Nursing Education articles published in 2020 suggests that a priori power analyses may be used infrequently in our community. Consequently, this column requested that authors conduct power analyses using increasingly rigorous standards (e.g., power levels) and report their findings well to readers. When a priori power analyses are missing or not reported clearly, reviewers and editors are encouraged to request authors ameliorate the shortfalls in their manuscripts. [ J Nurs Educ . 2021;60(11):605–606.]

This installment of the Methodology Corner raises the concern that our community may not be adequately attending to the threat missingness poses to valid nursing education research. To encourage the adoption of increasingly sound methodological practices, we highlight for our community that counts and relative frequencies and simple bivariate analyses are easily implemented tools that help formulate valid responses to missing data. [ J Nurs Educ . 2022;61(5):280–281.]

If left unattended, missingness can undermine the conclusions nurse researchers draw in their studies. Unfortunately, a cursory review of quantitative and mixed methods works recently published in the Journal of Nursing Education suggests missing data is left relatively unattended within our community. This column has recommended our researcher mitigate the emergence of missing data as much as possible, describe any missingness thoroughly, and adopt modern missing data techniques that help mitigate threats to validity introduced by missing data. Reviewers and editors are also encouraged to request from authors clear descriptions of the missingness in their studies as well as how they responded to any missingness. [ J Nurs Educ . 2022;61(2):65–66.]

Measures of parenting quality that are valid among high-risk families are needed for research and program evaluation. This study investigates the functioning of the Parent-Caregiver Involvement Scale -Short Form (P/CIS-SF) in a high-risk sample. The P/CIS-SF measures several behaviors pertaining to both "amount" and "quality," as well overall "impressions" of parenting. Participants were 170 six-month-olds and their mothers who were at risk for child abuse, impoverished, single, young, and had multiple children. Mother-infant interaction was videotaped in the home during semi-structured play. These high-risk parents scored in the midrange on most P/CIS-SF items, indicating that mothers engaged with their infants for approximately 50% of the semi-structured play session and the engagement tended to be of moderate quality. There were no floor or ceiling effects with the exception of "teaching behavior" which was rare among these parents. Analysis revealed that while "amount" and "quality" are interrelated, they capture distinct aspects of parenting. Validity of P/CIS-SF scores was indicated by correlation with Ainsworth's sensitivity scale and the Emotional Availability Scales intrusiveness subscale. P/CIS-SF scores also correlated with maternal education, but were independent of maternal risk (i.e., depression, drug use, domestic violence, and child abuse) within this high-risk sample. Implications are that the P/CIS-SF is valid for measuring parenting quality in high-risk families and is able to distinguish between dimensions of parenting, making it useful for targeted interventions.

A cursory look at the measurement practices in the Journal of Nursing Education revealed several deficits that our community is encouraged to address. In 2020, a little less than half of our quantitative studies did not provide reliability estimates from their own data, opting instead to provide estimates reported in the literature or none at all. Of the studies that did supply estimates using their data, only Cronbach's alpha was reported. Unfortunately, limitations with Cronbach's alpha and inconsistent reporting practices are likely undermining the science of nursing education. Researchers are encouraged to estimate reliability using their own data with increasingly valid techniques. [ J Nurs Educ . 2021;60(2):65–66.]

This article raises the concern that nursing education research may be unduly subject to several limitations inherited from an overreliance on Cronbach's α. Consequently, researchers are encouraged to make greater use of coefficient omega since it is expected to perform just as well as or better than Cronbach's α, especially when tau-equivalence goes unmet. Several resources are noted that may help researchers estimate omega for composite measurements. [ J Nurs Educ . 2021;60(8):429–430.]

Supplemental Digital Content is available in the text., Background: Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH, we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. Methods: We analyzed 13 037 participants enrolled in the NBR study (NIHR BioResource—Rare Diseases), of which 1148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension, we used the Bayesian rare variant association method BeviMed. Results: Heterozygous, high impact, likely loss-of-function variants in the kinase insert domain receptor (KDR) gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (posterior probability=0.989) and older age at diagnosis (posterior probability=0.912). We also provide evidence for familial segregation of a rare nonsense KDR variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the 5 patients harboring these predicted deleterious variants in KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions: The Bayesian inference approach allowed us to independently validate KDR, which encodes for the VEGFR2 (vascular endothelial growth factor receptor 2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for KDR being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.

We report a 2-year-old boy who was evaluated for difficult waking during prolonged intensive care unit admission associated with bone marrow transplant for Wiskott-Aldrich syndrome. Neurologic examination was found to be abnormal, with nuchal rigidity initially, then decreased extremity movement and areflexia developing over several days. Electromyogram showed length-dependent, axonal, sensorimotor polyneuropathy. Cerebrospinal fluid showed albuminocytologic dissociation suggestive of Guillain-Barre syndrome or acute motor and sensory axonal neuropathy variant. The patient was treated with immunotherapy and slowly showed signs of motor recovery over several months. A review of Wiskott-Aldrich syndrome, Guillain-Barre syndrome, immune-mediated complications of bone marrow transplantation, and acute weakness in the intensive care unit is provided in this case report.

Artificial neural networks (ANN) are increasingly applied to complex medical problem solving algorithms because their outcome prediction performance is superior to existing multiple regression models. ANN can successfully identify symptomatic cerebral vasospasm (SCV) in adults presenting after aneurysmal subarachnoid hemorrhage (aSAH). Although SCV is unusual in children with aSAH, the clinical consequences are severe. Consequently, reliable tools to predict patients at greatest risk for SCV may have significant value. We applied ANN modeling to a consecutive cohort of pediatric aSAH cases to assess its ability to predict SCV. A retrospective chart review was conducted to identify patients

The family Hepadnaviridae comprises small enveloped viruses with a partially double-stranded DNA genome of 3.0–3.4 kb. All family members express three sets of proteins (preC/C, polymerase and preS/S) and replication involves reverse transcription within nucleocapsids in the cytoplasm of hepatocytes. Hepadnaviruses are hepatotropic and infections may be transient or persistent. There are five genera: Parahepadnavirus, Metahepadnavirus, Herpetohepadnavirus, Avihepadnavirus and Orthohepadnavirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hepadnaviridae, which is available at ictv.global/report/hepadnaviridae.

Nursing researchers have been encouraged for decades to address the validity of their statistical conclusions in part by accumulating evidence from studies that are capable of correctly identifying relationships between variables when those relationships are truly present. However, it has been noted that only a fraction of nursing studies appear to have undertaken power analyses to help mitigate the risk of Type II statistical conclusion errors. This Methodology Corner article reviews some of the major aspects of power analysis procedures and reiterates recommendations that researchers who plan to use inferential statistical analyses also conduct a power analysis to guide decision making about target sample sizes to increase the validity of study findings. [ J Nurs Educ. 2018;57(5):262–264.]

BACKGROUND Obtaining high-quality images of cellular structures via immunofluorescence staining is critical for cellular localization studies. Often, these studies cannot be performed in parallel with certain oncology, virology, pharmacokinetic, and drug absorption studies due to model system technicalities requiring the cells to be cultured on porous membranes rather than glass or plastic. MATERIAL AND METHODS Here, we report a method of immunofluorescent staining of cells cultured on permeable membranes. RESULTS As proof of principle, HeLa cells grown on Transwell® membrane supports were stained with fluorescently labeled antibodies using this modified immunofluorescence staining method and visualized by fluorescent microscopy. CONCLUSIONS This protocol is a convenient alternative to staining cells on glass coverslips, thereby expanding the scope and applications of this important research tool.

The viroids of plants are the simplest known infectious genetic elements. They have RNA genomes of up to 400 nucleotides in length and no protein encoding capacity. Hepatitis delta virus (HDV), an infectious agent found only in humans co-infected with hepatitis B virus (HBV), is just slightly more complex, with an RNA genome of about 1700 nucleotides, and the ability to express just one small protein. Viroid and HDV RNAs share several features that include circular structure, compact folding, and replication via a rolling-circle mechanism. Both agents were detected because of their obvious pathogenic effects. Their simplicity demands a greater need than conventional RNA or DNA viruses to redirect host components for facilitating their infectious cycle, a need that directly and indirectly incites pathogenic effects. The mechanisms by which these pathogenic effects are produced are the topic of this review. In this context, RNA silencing mediates certain aspects of viroid pathogenesis., We apologize to colleagues whose work could not be cited for space limits. Work in RF laboratory has been supported by the Spanish Ministerio de Economia y Competitividad (grant BFU2014-56812-P).

Background: Rotational vertebral artery occlusion (RVAO) is associated with significant posterior circulation morbidity, recurrence, and lacks evidenced based treatment recommendations. Upper cervical spine abnormalities, lack of traumatic history, and dynamic vessel imaging aid in diagnosis and treatment options. Here, we evaluate our cohort of children with RVAO for associated musculoskeletal, angiographic, and arterial tortuosity factors in light of management strategies and outcomes. Methods: Five pediatric patients with RVAO were evaluated with dynamic arterial compression with either CT based angiography or digital subtraction angiography. Solitary factors such as ponticulous posticus, hypermobility comorbidities, 3D-print models and arterial tortuosity index were examined in relation to management strategies. Results: Musculoskeletal abnormalities were associated in all cases presenting with evidence of dynamic angiographic compression. Not all cases had evidence angiographic compression. A mild elevation in arterial tortuosity index was observed. All five patients were managed with treatment approaches including antiplatelet/coagulation therapies, vertebral artery decompression, and/or posterior cervical fusion. Conclusion: Treatment strategies relied on identifiable compression factors associated with the level of vertebral artery dissection. In children, upper cervical bony abnormalities and dynamic compression at the VA3 level are common to RVAO diagnosis. Further examination of arterial tortuosity as an index in understanding the susceptibility of children towards RVAO is required.

Although the 10-item Perceived Stress Scale (PSS-10) is a popular measure, a review of the literature reveals 3 significant gaps: (a) There is some debate as to whether a 1- or a 2-factor model best describes the relationships among the PSS-10 items, (b) little information is available on the performance of the items on the scale, and (c) it is unclear whether PSS-10 scores are subject to gender bias. These gaps were addressed in this study using a sample of 1,236 adults from the National Survey of Midlife Development in the United States II. Based on self-identification, participants were 56.31% female, 77% White, 17.31% Black and/or African American, and the average age was 54.48 years (SD = 11.69). Findings from an ordinal confirmatory factor analysis suggested the relationships among the items are best described by an oblique 2-factor model. Item analysis using the graded response model provided no evidence of item misfit and indicated both subscales have a wide estimation range. Although t tests revealed a significant difference between the means of males and females on the Perceived Helplessness Subscale (t = 4.001, df = 1234, p < .001), measurement invariance tests suggest that PSS-10 scores may not be substantially affected by gender bias. Overall, the findings suggest that inferences made using PSS-10 scores are valid. However, this study calls into question inferences where the multidimensionality of the PSS-10 is ignored.

Apoptosis serves as a powerful defense against damaged or pathogen-infected cells. Since apoptosis is an effective defense against viral infection, many viruses including poxviruses, encode proteins to prevent or delay apoptosis. Here we show that ectromelia virus, the causative agent of mousepox encodes an anti-apoptotic protein EVM025. Here we demonstrate that expression of functional EVM025 is crucial to prevent apoptosis triggered by virus infection and staurosporine. We demonstrate that the expression of EVM025 prevents the conformational activation of the pro-apoptotic proteins Bak and Bax, allowing the maintenance of mitochondrial membrane integrity upon infection with ECTV. Additionally, EVM025 interacted with intracellular Bak. We were able to demonstrate that EVM025 ability to inhibit Bax activation is a function of its ability to inhibit the activity of an upstream BH3 only protein Bim. Collectively, our data indicates that EVM025 inhibits apoptosis by sequestering Bak and inhibiting the activity of Bak and Bax.

Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent–child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder. We searched for variants in 31 genes linked to GPI-anchor biogenesis and detected rare biallelic variants in PGAP3, PIGN, PIGT (n=2), PIGO and PIGL, providing a likely diagnosis for six families. In five families, the variants were in a compound heterozygous configuration while in a consanguineous Afghani kindred, a homozygous c.709G>C; p.(E237Q) variant in PIGT was identified within 10–12 Mb of autozygosity. Validation and segregation analysis was performed using Sanger sequencing. Across the six families, five siblings were available for testing and in all cases variants co-segregated consistent with them being causative. In four families, abnormal alkaline phosphatase results were observed in the direction expected. FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the variants in PIGN, PIGT and PIGO all led to reduced activity. Splicing assays, performed using leucocyte RNA, showed that a c.336-2A>G variant in PIGL resulted in exon skipping and p.D113fs*2. Our results strengthen recently reported disease associations, suggest that defective GPI-anchor biogenesis may explain ~0.15% of individuals with developmental disorders and highlight the benefits of data sharing.

Background/Hypothesis: Stroke in children ≤18 years is an important public health problem. The incidence of stroke in adults has been falling over several decades, likely attributable to better management of vascular risk factors. Children have different risk factors for cerebrovascular disease, however we wanted to see if similar trends were observed in pediatric stroke. We examined pediatric hospital discharges for stroke over the last decade. Methods: We used the Kids’ Inpatient Database (KID) for 2000, 2003, 2006, and 2009 using the published weighting guidelines to estimate the number of overall discharges and number of discharges due to stroke. To identify stroke we queried primary position discharge ICD-9 stroke codes 430, 431, 432.9, 434.X1, 434.9, and 435.9; previously identified to have high PPV for detection of stroke in children in a physician-verified epidemiology stroke study. We calculated incidence for stroke among hospitalized children as number of stroke discharges / total number of discharges, and expressed the ratio per 100,000 discharges. We compared trends in stroke type, age, race and sex over the decade. Results: Stroke incidence in 2000 was 26.4 (95% CI 23.2-29.6). In the 2009 sample, incidence was 30.9 (27.7-34.1) showing a non-significant increasing trend over time. Ischemic stroke was increasingly diagnosed over the same interval with a statistically significantly higher incidence in 2006 and 2009 compared to 2000 (Figure 1). Groups with highest stroke incidence in hospitalized children were 10-14 year olds, males, and children identifying with the racial group “black.” Conclusion: In the KID database, we identified a rising incidence of ischemic stroke diagnosis over the past decade, however the overall rate of pediatric stroke has not increased significantly. The increasing rate of an ischemic stroke discharge diagnosis may be the result of increased awareness of childhood stroke.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark ( Squalus acanthias) and the sea lamprey ( Petromyzon marinus) , exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

The 195 amino acid basic protein (δAg) of hepatitis delta virus (HDV) is essential for replication of the HDV RNA genome. Numerous properties have been mapped to full-length δAg and attempts made to link these to secondary, tertiary and quaternary structures. Here, for the full-size δAg, extensive intrinsic disorder was predicted using PONDR-FIT, a meta-predictor of intrinsic disorder, and evidenced by circular dichroism measurements. Most δAg amino acids are in disordered configurations with no more than 30% adopting an α-helical structure. In addition, dynamic light scattering studies indicated that purified δAg assembled into structures of as large as dodecamers. Cross-linking followed by denaturing polyacrylamide gel electrophoresis revealed hexamers to octamers for this purified δAg and at least this size for δAg found in virus-like particles. Oligomers of purified δAg were resistant to elevated NaCl and urea concentrations, and bound without specificity to RNA and single- and double-stranded DNAs.

The recent H1N1 epidemic has resulted in a large number of deaths, primarily from acute hypoxemic respiratory failure. We reviewed the current strategies to rescue patients with severe hypoxemia. Included in these strategies are high-frequency oscillatory ventilation, airway pressure release ventilation, inhaled vasodilators, and the use of extracorporeal life support. All of these strategies are targeted at improving oxygenation, but improved oxygenation alone has yet to be demonstrated to correlate with improved survival. The risks and benefits of these strategies, including cost-effectiveness data, are discussed.

This article addresses some of the questions relating to how hepatitis δ virus (HDV), an agent so far unique in the animal world, might have arisen. HDV was discovered in patients infected with hepatitis B virus (HBV). It generally makes HBV infections more damaging to the liver. It is a subviral satellite agent that depends upon HBV envelope proteins for its assembly and ability to infect new cells. In other aspects of replication, HDV is both independent of and very different from HBV. In addition, the small single-stranded circular RNA genome of HDV, and its mechanism of replication, demonstrate an increasing number of similarities to the viroids – a large family of helper-independent subviral agents that cause pathogenesis in plants.

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (δAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that δAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, δAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of δAg in the presence and absence of replicating and nonreplicating HDV RNAs. When δAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both δAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, δAg moved to the nucleoplasm, but nucleolin was unchanged. When δAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of δAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of δAg altered at specific locations considered to be essential for protein function. These studies provide evidence that δAg does not interact directly with either Pol II or nucleolin and that forms of δAg which support replication are also capable of prior nucleolar transit.

Article-at-a-Glance Background Implementation of evidence-based quality improvement (QI) initiatives is not without its challenges. Recent experience in the design, implementation, and evaluation of three QI initiatives at the University of California, San Francisco Medical Center (UCSF) suggests lessons learned that may be generalizable to other QI initiatives. Initiatives Between December 2002 and May 2006, a ventilator bundle of care and a tight glycemic control (TGC) protocol were implemented in the intensive care units (ICUs), and early goal-directed therapy (EGDT) for patients with severe sepsis or septic shock was implemented in the ICUs and emergency department. The initiatives were selected on the basis of the magnitude of the problem, strength of the evidence regarding associated reductions in morbidity and mortality in the critically ill, and cost-effectiveness. Lessons Learned A number of challenges in QI processes and strategies for success were identified via retrospective analysis within the construct of the Plan-Do-Study-Act model, representing a novel use of the model. Pitfalls most commonly occurred in the planning stage. Suggested strategies for success include using an interdisciplinary team, selecting a champion, securing additional resources, identifying specific goals and providing feedback on progress, using work-flow analyses and stepwise implementation and/or pilot testing, creating standard work, eliciting feedback from staff, and celebrating successes. The knowledge gained from these initiatives has been disseminated at UCSF, and the initiatives have helped to raise general awareness regarding the importance of quality. Conclusions The ventilator bundle of care, TGC, and EGDT are still in use at UCSF, with modification of the initiatives occurring as new evidence becomes available.

A universal cellular defense mechanism against viral invasion is the elimination of infected cells through apoptotic cell death. To counteract host defenses many viruses have evolved complex apoptosis evasion strategies. The oncogenic human retrovirus HTLV-1 is the etiological agent of adult-T-cell leukemia/lymphoma (ATLL) and the neurodegenerative disease known as HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The poor prognosis in HTLV-1-induced ATLL is linked to the resistance of neoplastic T cells against conventional therapies and the immuno-compromised state of patients. Nevertheless, several studies have shown that the apoptotic pathway is largely intact and can be reactivated in ATLL tumor cells to induce specific killing. A better understanding of the molecular mechanisms employed by HTLV-1 to counteract cellular death pathways remains an important challenge for future therapies and the treatment of HTLV-1-associated diseases.

This study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated by the transfection of 293T cells with three plasmids: (i) a human immunodeficiency virus (HIV) packaging construct, (ii) a transfer plasmid expressing a reporter gene, and (iii) a plasmid expressing large (L), middle (M), and small (S) HBV envelope proteins. After 2 days, hepatitis B surface antigen and the antigenic forms of L, M, and S were detected at the cell surface by flow cytometry. Also, virus particles that were able to infect cultured primary human hepatocytes (PHH) were released. Under optimal conditions, 50% of PHH could be infected. In addition, the susceptibility of PHH and the resistance of other cell types to the pseudotype particles were similar to those observed for HBV and hepatitis delta virus (HDV), which shares the same L, M, and S. Furthermore, the infection of PHH by the pseudotype was sensitive to known inhibitors of HBV and HDV entry. These findings of specific and efficient infection of hepatocytes could be applicable to liver-specific gene therapy and may help clarify the attachment and entry mechanism used by HBV and HDV.

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.

RNA-directed DNA synthesis by detergent-disrupted virions of Rous sarcoma virus (RSV) initiates by the covalent attachment of pdA to the 3'-terminal rA of a 4S RNA hydrogen-bonded to the 70S RNA template. This 4S "primer" has structural features of tRNA and can be aminoacylated with methionine. Synthesis and integration of provirus DNA can be monitored in both permissive (duck) and nonpermissive (mouse) cells acutely infected with RSV. The results of these studies, as well as data obtained with RSV-infected mammalian cells which have reverted from a transformed to a pheno-typically normal state, indicate that integration of viral genes into the host chromosome is not sufficient cause for transformation. Pertinent features of virus-specific RNA-directed DNA synthesis in vitro and in vivo are reviewed and compared.

Vaccinia virus, the prototypic member of the orthopoxvirus genus, encodes the mitochondrial-localized protein F1L that functions to protect cells from apoptotic death and inhibits cytochrome c release. We previously showed that F1L interacts with the pro-apoptotic Bcl-2 family member Bak and inhibits activation of Bak following an apoptotic stimulus (Wasilenko, S. T., Banadyga, L., Bond, D., and Barry, M. (2005) J. Virol. 79, 14031-14043). In addition to Bak, the pro-apoptotic protein Bax is also capable of initiating cytochrome c release suggesting that vaccinia virus infection could also inhibit Bax activity. Here we show that F1L inhibits the activity of the pro-apoptotic protein Bax by inhibiting oligomerization and N-terminal activation of Bax. F1L expression also inhibited the subcellular redistribution of Bax to the mitochondria and the insertion of Bax into the outer mitochondrial membrane. The ability of F1L to inhibit Bax activation does not require Bak, because F1L expression inhibited cytochrome c release and Bax activation in Bak-deficient cells. No interaction between Bax and F1L was detected during infection, suggesting that F1L functions upstream of Bax activation. Notably, F1L was capable of interacting with the BH3-only protein BimL as shown by co-immunoprecipitation, and F1L expression inhibited apoptosis induced by BimL. These studies suggest that, in addition to interacting with the pro-apoptotic protein Bak, F1L also functions to indirectly inhibit the activation of Bax, likely by interfering with the pro-apoptotic activity of BH3-only proteins such as BimL.

To review common and life-threatening complications resulting in intensive care unit admission for patients undergoing orthopedic surgery procedures. To identify specific diagnostic techniques and treatment modalities that may improve the outcome of critically ill orthopedic surgery patients.A review of the current literature regarding the care of orthopedic surgery patients was performed.Orthopedic surgery patients are vulnerable to a number of complications, particularly pulmonary complications related to scoliosis surgery, embolic complications of joint arthroplasty, and complications related to the use of opioids and sedatives in an elderly population. They also are susceptible to transfusion-related complications such as transfusion-related acute lung injury and transfusion-associated circulatory overload. Specific strategies for management of these complex patients are identified.Orthopedic surgery patients represent a significant and growing proportion of patients in intensive care units. They develop unique complications, and management of these complications requires understanding of preoperative co-morbidities, intraoperative management, and early recognition and treatment of postoperative complications. Prevention and early identification of these complications are the most efficacious routes to improving outcomes in this patient population.