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Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N -methyltransferase 2E-antisense 1 ( KMT2E-AS1 ) and histone lysine N-methyltransferase 2E ( KMT2E ). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1 /KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

Background: Left heart disease is the most common cause of pulmonary hypertension (PH) and is frequently accompanied by increases in pulmonary vascular resistance. However, the distinction between phenotypes of PH due to left heart disease with a normal or elevated pulmonary vascular resistance-isolated postcapillary PH (IpcPH) and combined pre- and postcapillary PH (CpcPH), respectively-has been incompletely defined using unbiased methods., Methods and Results: Patients with extremes of IpcPH versus CpcPH were identified from a single-center record of those who underwent right heart catheterization. Individuals with left ventricular ejection fraction <40% or with potential causes of PH beyond left heart disease were excluded. Medication usage in IpcPH and CpcPH was compared across Anatomical Therapeutic Chemical classes and identified vitamin K antagonists as the only medication with pharmacome-wide significance, being more commonly used in CpcPH and for an indication of atrial fibrillation in ≈90% of instances. Accordingly, atrial fibrillation prevalence was significantly higher in CpcPH in a phenome-wide analysis. Review of echocardiographic data most proximal to right heart catheterization revealed that left atrial diameter indexed to body surface area-known to be associated with atrial fibrillation-was increased in CpcPH regardless of the presence of atrial fibrillation. An independent cohort with serial right heart catheterizations and PH-left heart disease showed a significant positive correlation between change in left atrial diameter indexed to body surface area and change in pulmonary vascular resistance., Conclusions: Guided by pharmacomic and phenomic screens in a rigorously phenotyped cohort, we identify a longitudinal association between left atrial diameter indexed to body surface area and pulmonary vascular resistance with implications for the future development of diagnostic, prognostic, and therapeutic tools.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium ( r 2 >0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

Quantitative methods have thus far been the predominant methodological stance of virtual presence research, leaving much to be desired in terms of qualitative understanding. Yet, virtual experiences are a highly personal engagement, unique to each individual, and their presence in virtual reality can be viewed in terms of its experiential individuality. This aspect of the virtual experience is overlooked by conventional quantitative methods, which clusters ratings or scores to form group deductions. Therefore, to address the qualitative gap in the literature and provide an appropriate examination of virtual experiences from the perspective of the individual, an Interpretative Phenomenological Approach was undertaken. This alternate methodology sought to reveal which aspects of virtual experiences users identify as enabling feelings of presence. Examination of common themes among accounts of individuals were performed, to investigate the generation of feelings of presence in virtual reality. Online recruitment provided six interviewees who participated in online semi-structured interviews, prior to Interpretive Phenomenological Analysis. Three superordinate themes were identified: visual satisfaction, freedom of interaction and suspension of real life. Expectance, realism and prevention of disbelief are among the sub-themes identified that contributed to the interviewee's highly present experiences. The identified themes demonstrated the greatest influences of enabling a deeper sense of presence, in turn enhancing their experiences within virtual reality. In acknowledging these mitigating influences, it is hoped this may enable future virtual systems to build upon the research provided and produce consistently high-presence experiences. Consequently, this can aid educational, therapeutic and entertainment applications of virtual reality., Competing Interests: Conflict of interestNo conflicts of interest are declared., (© The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: The prognostic impact of tumor-infiltrating lymphocytes (TILs) on outcomes and treatment efficacy for patients with melanoma in the contemporary era remains poorly characterized., Methods: Consecutive patients who underwent wide excision and sentinel lymph node biopsy for cutaneous melanoma 1 mm thick or thicker at a single institution were identified (2006-2019). The patients were stratified based on primary tumor TIL status as brisk (bTILs), non-brisk (nbTILs), or absent (aTILs). Associations between patient factors and outcomes were analyzed using multivariable analysis., Results: Of the 1017 patients evaluated, 846 (83.2 %) had primary TILs [nbTILs (n = 759, 89.7 %) and bTILs (n = 87, 10.3 %)]. In the multivariable analysis, the patients with any type of TILs had higher rates of regression [odds ratio (OR), 1.86; p = 0.016], lower rates of acral lentiginous histology (OR, 0.22; p < 0.001), and lower rates of SLN positivity (OR, 0.64; p = 0.042) than those without TILs. The multivariable analysis found no association between disease-specific survival and bTILs [hazard ratio (HR), 1.04; p = 0.927] or nbTILs (HR, 0.89; p = 0.683). An association was found between bTILs and recurrence-free survival (RFS) advantage [bTILs (HR 0.46; p = 0.047), nbTILs (HR 0.71; p = 0.088)], with 5-year RFS rates of 84 % for bTILs, 71.8 % for nbTILs, and 68.4 % for aTILs (p = 0.044). For the 114 immune checkpoint blockade (ICB)-naïve patients who experienced a recurrence treated with ICB therapy, no association was observed between progression-free survival and bTILs (HR, 0.64; p = 0.482) or nbTILs (HR, 0.58; p = 0.176)., Conclusions: The prognostic significance of primary TILs in the contemporary melanoma era appears complex. Further studies characterizing the phenotype of TILs and their association with regional metastasis and responsiveness to ICB therapy are warranted., (© 2022. Society of Surgical Oncology.)

Pulmonary arterial hypertension (PAH) is an enigmatic and deadly vascular disease with no known cure. Recent years have seen rapid advances in our understanding of the molecular underpinnings of PAH, with an expanding knowledge of the molecular, cellular, and systems-level drivers of disease that are being translated into novel therapeutic modalities. Simultaneous advances in clinical technology have led to a growing list of tools with potential application to diagnosis and phenotyping. Guided by fundamental biology, these developments hold the potential to usher in a new era of personalized medicine in PAH with broad implications for patient management and great promise for improved outcomes.

Utilizing publicly available ribonucleic acid sequencing data, we identified SCUBE1 as a BMPR2-related gene differentially expressed between induced pluripotent stem cell-endothelial cells derived from pulmonary arterial hypertension (PAH) patients carrying pathogenic BMPR2 mutations and control patients without mutations. Endothelial SCUBE1 expression was decreased by known triggers of PAH, and its down-regulation recapitulated known BMPR2-associated endothelial pathophenotypes in vitro. Meanwhile, SCUBE1 concentrations were reduced in plasma obtained from PAH rodent models and patients with PAH, whereas plasma concentrations were tightly correlated with hemodynamic markers of disease severity. Taken together, these data implicate SCUBE1 as a novel contributor to PAH pathogenesis with potential therapeutic, diagnostic, and prognostic applications., Competing Interests: This work was supported by National Institutes of Health grants R01 HL124021, HL 122596, HL 138437, and UH2 TR002073 (to Dr. Chan); 2P01 HL103455 and 1R01AG058659 (to Dr. Simon); HL142084 and HL136143 (to Dr. Lee); R00HL121076-03, R01HL143967, and R01HL142629 (to Dr. Dutta); and 5T32HL129964-04 (to Dr. Sun). This work was also supported by American Heart Association Established Investigator Award 18EIA33900027 (to Dr. Chan), as well as Texas A and M University Systems Chancellors’ Research Initiative for Center for Computational Systems Biology at Prairie View A and M University (to Dr. Kim). Drs. Sun and Chan filed patent applications regarding the targeting of bone morphogenetic protein signaling in pulmonary hypertension. Dr. Simon has received research grants from Novartis and Aadi; and has received consulting fees from Complexa, United Therapeutics, and Actelion. Dr. Mazurek has served as a consultant for United Therapeutics and Actelion; and has received research support from Tenax Therapeutics, Complexa Inc., and Actelion. Dr. Vaidya has served as a consultant for Bayer, Actelion, and United Therapeutics. Dr. Chan has served as a consultant for Zogenix, Aerpio, and United Therapeutics; is a director, officer, and shareholder in Numa Therapeutics; and has held research grants from Actelion and Pfizer. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Hypoxia is a driver of cell movement in processes such as development and tumor progression. The cellular response to hypoxia involves a transcriptional program mediated by hypoxia-inducible factors, but translational control has emerged as a significant contributor. In this study, we demonstrate that a cell-cell adhesion molecule, cadherin-22, is upregulated in hypoxia via mTORC1-independent translational control by the initiation factor eIF4E2. We identify new functions of cadherin-22 as a hypoxia-specific cell-surface molecule involved in cancer cell migration, invasion and adhesion. Silencing eIF4E2 or cadherin-22 significantly impaired MDA-MB-231 breast carcinoma and U87MG glioblastoma cell migration and invasion only in hypoxia, while reintroduction of the respective exogenous gene restored the normal phenotype. Cadherin-22 was evenly distributed throughout spheroids and required for their formation and support of a hypoxic core. Conversely, E-cadherin translation was repressed by hypoxia and only expressed in the oxygenated cells of U87MG spheroids. Furthermore, immunofluorescence on paraffin-embedded human tissue from 40 glioma and 40 invasive ductal breast carcinoma patient specimens revealed that cadherin-22 expression colocalized with areas of hypoxia and significantly correlated with tumor grade and progression-free survival or stage and tumor size, respectively. This study broadens our understanding of tumor progression and metastasis by highlighting cadherin-22 as a potential new target of cancer therapy to disable hypoxic cancer cell motility and adhesion.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke-induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 μm) and CBA/J was the least susceptible (Δ CL -0.3 ± 1.2 μm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Rationale: Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD)., Objectives: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer., Methods: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and Kras LSL/G12D mouse models of lung cancer., Measurements and Main Results: We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer., Conclusions: The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

Rationale: Genetic association studies in chronic obstructive pulmonary disease have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. Because rare variation is also likely to contribute to susceptibility, we used whole-genome sequencing of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to chronic obstructive pulmonary disease susceptibility., Objectives: To identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction., Methods: We identified heavy smokers that were resistant (n = 65) or susceptible (n = 64) to emphysema with severe airflow obstruction in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We filtered whole-genome sequencing results to include only rare variants and conducted single variant tests, region-based tests across the genome, gene-based tests, and exome-wide tests., Measurements and Main Results: We identified several suggestive associations with emphysema with severe airflow obstruction, including a suggestive association of all rare variation in a region within the gene ZNF816 (19q13.41; P = 4.5 × 10 -6 ), and a suggestive association of nonsynonymous coding rare variation in the gene PTPRO (P = 4.0 × 10 -5 ). Association of rs61754411, a rare nonsynonymous variant in PTPRO, with emphysema and obstruction was demonstrated in all non-Hispanic white individuals in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We found that cells containing this variant have decreased signaling in cellular pathways necessary for survival and proliferation., Conclusions: PTPRO is a novel candidate gene in emphysema with severe airflow obstruction, and rs61754411 is a previously unreported rare variant contributing to emphysema susceptibility. Other suggestive candidate genes, such as ZNF816, are of interest for future studies.

Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH-HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH-HFpEF. To develop a mouse model of PH-HFpEF, we exposed 36 mouse strains to 20 weeks of high-fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure-volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH-HFpEF. Of the strains that develop PH-HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD-induced metabolic syndrome and had low variability in hemodynamics. HFD-treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end-diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end-diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD-induced PH-HFpEF. These studies validate an HFD/AKR/J mouse model of PH-HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high-fat diet (HFD) in some inbred mouse strains. We conducted a genome-wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD-induced PH. Mice from 36 inbred and wild-derived strains were fed with regular diet or HFD for 20 weeks beginning at 6-12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end-systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest-path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH-related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high-fat feeding. Network-based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest-path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high-fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network-based approach adds evidence for a role for Egfr in murine PH.

The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton-based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.

Cigarette smoke is the most common cause of pulmonary emphysema, which results in an irreversible loss of lung structure and function. Th1 and Th17 immune responses have been implicated in emphysema pathogenesis; however, the drivers of emphysema-associated immune dysfunction are not fully understood. In this issue of the JCI, Shan and colleagues found that peroxisome proliferator-activated receptor γ (PPARγ) is downregulated in APCs isolated from the lungs of emphysematous chronic smokers and mice exposed to cigarette smoke. Furthermore, treatment with a PPARγ agonist prevented emphysema development and appeared to reduce emphysema-associated lung volume expansion in mice exposed to cigarette smoke. Further work will need to be done to evaluate the potential of PPARγ agonists to restore lung capacity in emphysematous patients.

Glutathione peroxidase-1 (GPx-1) is a crucial antioxidant enzyme, the deficiency of which promotes atherogenesis. Accordingly, we examined the mechanisms by which GPx-1 deficiency enhances endothelial cell activation and inflammation. In human microvascular endothelial cells, we found that GPx-1 deficiency augments intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by redox-dependent mechanisms that involve NFκB. Suppression of GPx-1 enhanced TNF-α-induced ROS production and ICAM-1 expression, whereas overexpression of GPx-1 attenuated these TNF-α-mediated responses. GPx-1 deficiency prolonged TNF-α-induced IκBα degradation and activation of ERK1/2 and JNK. JNK or NFκB inhibition attenuated TNF-α induction of ICAM-1 and VCAM-1 expression in GPx-1-deficient and control cells, whereas ERK1/2 inhibition attenuated only VCAM-1 expression. To analyze further signaling pathways involved in GPx-1-mediated protection from TNF-α-induced ROS, we performed microarray analysis of human microvascular endothelial cells treated with TNF-α in the presence and absence of GPx-1. Among the genes whose expression changed significantly, dual specificity phosphatase 4 (DUSP4), encoding an antagonist of MAPK signaling, was down-regulated by GPx-1 suppression. Targeted DUSP4 knockdown enhanced TNF-α-mediated ERK1/2 pathway activation and resulted in increased adhesion molecule expression, indicating that GPx-1 deficiency may augment TNF-α-mediated events, in part, by regulating DUSP4.

The IkappaB kinase (IKK) complex consists of the catalytic subunits IKKalpha and IKKbeta and a regulatory subunit, IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKalpha, but not IKKbeta, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKalpha in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKalpha knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKalpha and E2F1, and this interaction occurred on promoters bound by E2F1. IKKalpha also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKalpha can influence estrogen-mediated cell cycle progression through its regulation of E2F1.

Human immunodeficiency virus type 1 (HIV-1) selects a host cell tRNA as the primer for the initiation of reverse transcription. In a previous study, transport of the intact tRNA from the nucleus to the cytoplasm during tRNA biogenesis was shown to be a requirement for the selection of the tRNA primer by HIV-1. To further examine the importance of tRNA structure for transport and the selection of the primer, yeast tRNA(Phe) mutants were designed such that the native tRNA structure would be disrupted to various extents. The capacity of the mutant tRNA(Phe) to complement a defective HIV-1 provirus that relies on the expression of yeast tRNA(Phe) for infectivity was determined. We found a direct relationship between intact tRNA conformation and the capacity to be selected by HIV-1 for use in reverse transcription. tRNA(Phe) mutants that retained the capacity for nucleocytoplasmic transport, indicative of overall intact conformation, complemented the defective provirus. The mutant tRNAs were not aminoacylated, and the levels of complementation were lower than that for wild-type tRNA(Phe), which did undergo transport and aminoacylation. Taken together, these results demonstrate that HIV-1 primer selection is most dependent on a tRNA structure necessary for nucleocytoplasmic transport, consistent with primer selection occurring in the cytoplasm at or near the site of protein synthesis.

The replication in human peripheral blood mononuclear cells (PBMC) of unique HIV-1 that select tRNA(His) or tRNA(Lys1,2) for reverse transcription was compared to the wild-type virus that uses tRNA(Lys,3). HIV-1 with only the primer-binding site (PBS) changed to be complementary to these alternative tRNAs initially replicated more slowly than the wild-type virus in PBMC, although all viruses eventually reached equivalent growth as measured by p24 antigen. Viruses with only a PBS complementary to the 3' terminal 18 nucleotides of tRNA(His) or tRNA(Lys1,2) reverted to use tRNA(Lys3). HIV-1 with mutations in the U5-PBS to allow selection of tRNA(His) and tRNA(Lys1,2) following long-term growth in SupT1 cells were also evaluated for growth and PBS stability following replication in PBMC. Although both viruses initially grew slower than wild type, they maintained a PBS complementary to the starting tRNA and did not revert to the wild-type PBS after long-term culture in PBMC. Analysis of the U5-PBS regions following long-term culture in PBMC also revealed few changes from the starting sequences. The virus that stably used tRNA(His) was less infectious than the wild type. In contrast, the virus that stably used tRNA(Lys1,2) evolved to be as infectious as wild-type virus following extended culture in PBMC. The results of these studies highlight the impact of the host cell on the tRNA primer selection process and subsequent infectivity of HIV-1.

All naturally occurring human immune deficiency viruses (HIV-1) select and use tRNA(Lys,3) as the primer for reverse transcription. Studies to elucidate the mechanism of tRNA selection from the intracellular milieu have been hampered due to the difficulties in manipulating the endogenous levels of tRNA(Lys,3). We have previously described a mutant HIV-1 with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe) that relies on transfection of yeast tRNA(Phe) for infectivity. To more accurately recapitulate the selection process, a cDNA was designed for the intracellular expression of the yeast tRNA(Phe). Increasing amounts of the plasmid encoding tRNA(Phe) resulted in a corresponding increase in levels of yeast tRNA(Phe) in the cell. The yeast tRNA(Phe) isolated from cells transfected with the cDNA for yeast tRNA(Phe), or in the cell lines expressing yeast tRNA(Phe), were aminoacylated, indicating that the expressed yeast tRNA(Phe) was incorporated into tRNA biogenesis pathways and translation. Increasing the cytoplasmic levels of tRNA(Phe) resulted in increased encapsidation of tRNA(Phe) in viruses with a PBS complementary to tRNA(Phe) (psHIV-Phe) or tRNA(Lys,3) (wild-type HIV-1). Production of infectious psHIV-Phe was dependent on the amount of cotransfected tRNA(Phe) cDNA. Increasing amounts of plasmids encoding yeast tRNA(Phe) produced an increase of infectious psHIV-Phe that plateaued at a level lower than that from the transfection of the wild-type genome, which uses tRNA(Lys,3) as the primer for reverse transcription. Cell lines were generated that expressed yeast tRNA(Phe) at levels approximately 0.1% of that for tRNA(Lys,3). Even with this reduced level of yeast tRNA(Phe), the cell lines complemented psHIV-Phe over background levels. The results of these studies demonstrate that intracellular levels of primer tRNA can have a direct effect on HIV-1 infectivity and further support the role for PBS-tRNA complementarity in the primer selection process.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3'-terminal nucleotides of tRNA(3)(Lys). Why all known strains of HIV-1 select tRNA(3)(Lys) for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNA(Glu). Since the virus was not initially designed to use tRNA(Glu), the virus had selected tRNA(Glu) from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNA(Glu) may provide new insights into the preference for tRNA(3)(Lys). HIV-1 constructed with the PBS complementary to tRNA(Glu) was more stable than HIV-1 with the PBS complementary to tRNA(Met) or tRNA(His); however, all of these viruses eventually reverted back to using tRNA(3)(Lys) following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNA(Glu) remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA(3)(Lys) are exploited by HIV-1 for selection as the reverse transcription primer.

Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNA(Phe) to be supplied in trans for infectivity. Wild-type yeast tRNA(Phe) expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNA(Phe) without the D loop (tRNA(Phe)D(-)) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNA(Phe)D(-) was directly transfected into the cytoplasm of cells. A tRNA(Phe) mutant (tRNA(Phe)UUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNA(Phe). Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.

The Mycobacterium avium complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the M. avium complex. We previously described the ser2 gene cluster, encoding the synthesis of the haptenic oligosaccharide (2, 3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (ser2A) encoding a putative rhamnosyltransferase. Sequencing of the ser2A locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in Mycobacterium smegmatis, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed rtfA, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of rtfA will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple M. avium complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.

The explosive development of minimally invasive surgery has had a staggering impact on the hospital, operating room, and surgeon, as well as on the medical equipment industry and insurance carriers. As a result of (1) the overwhelming demand by the public, (2) the potential of future developments of this modality, and (3) the progressive geometric influence that has spread to the various surgical subspecialties, unprecedented pressure has been placed on our systems for training, credentialing, developing, supplying, and evaluating changes in surgical technique.

Twenty-two women with on average more than 3 years unexplained infertility including normal menstrual cycles were studied because of borderline hyperprolactinemia (repeated serum prolactin concentrations 400-1000 mU/l). Randomized double-blind treatment with bromocriptine 5 mg daily (leading to adequate prolactin suppression) or matching placebo, each for 4 cycles, resulted in a cumulative rate after 4 months of 14.3 +/- 10.6% (SE) in both groups. Also, the results of treatment were not significantly related to the serum prolactin response to an intravenous injection of thyrotropin releasing hormone, whether the response was blunted (ratio of peak to basal prolactin within 60 minutes of injection no greater than 2), intermediate (greater than 2 to 4) or normal (greater than 4).

Basal serum prolactin concentrations were compared with mid-luteal serum progesterone concentrations and prospectively studied conception rates up to 12 months in women with otherwise unexplained infertility including a normal menstrual cycle. There were altogether 188 women with mean prolactin levels up to 3075 mU/l: 47 with prolactin up to 200 mU/l, 100 with prolactin 201-400 mU/l, 18 up to 600 mU/l, 16 up to 800 mU/l, and 7 greater than 800 mU/l. There were no significant differences in the mean progesterone values or chance of conception between any of the groups. The findings indicate that prolactin measurement is generally of no value in normally menstruating women, except possibly in the unusual event of evidence of persistent distinct luteal deficiency (not found in the present study).

Artificial insemination with husband's semen into the cervical canal and uterine cavity (high AIH) was assessed by a randomized controlled prospective study in 46 couples whose infertility was due to failure of sperm mucus penetration, as defined by negative postcoital tests, after excluding all interfering female factors and men with sperm density less than 1 x 10(6)/ml. Seminal analysis was abnormal in 18 of the 46 men and sperm antibodies in semen were detected in 19 of the remaining 28 with normal seminal analysis. Overall, the cumulative conception rate after 6 months with AIH was 4.7 (SE 3.8)% and without treatment was 6.6 (SE 3.9)%. The results were unaffected by the findings on seminal analysis or by the presence or absence of sperm antibodies in semen. AIH appeared to be of no benefit.

A mid-luteal serum progesterone measurement was made in 579 cycles from 159 infertile women with unexplained infertility and the results compared with those in 267 cycles from 58 normal controls. There were no significant differences between the two groups in terms of mean progesterone values (41 versus 44 nmol/l); incidence of defective cycles (progesterone less than 28 nmol/l: 17 versus 15%) or severely defective cycles (progesterone less than 14 nmol/l: 4 versus 3%); incidence of a clinical diagnosis of luteal deficiency (LD) (at least two out of three cycles defective: 13 versus 12%); or incidence of persistent LD (continuing into a second series of two to three cycles: 1.7 versus 4.9%). There were no differences related to age, previous pregnancy or duration of infertility. These findings suggest that defective cycles occur as a random phenomenon in infertility and no more often than in normal women, and a diagnosis of LD implying a persistent condition to explain prolonged infertility in women with normal menstrual cycles must be a rare entity and requires at least six cycles of investigation (or none!) before treatment is considered.

Infertile women without any inherent female infertility factors and able to secrete normal cervical mucus were studied prospectively in relation to post-coital sperm-mucus penetration (PCT) and their partner's seminal analysis, excluding men with azoospermia. Time-specific cumulative conception rates calculated as for life-table analysis were related to each measured seminal variable on routine analysis of 2-3 samples (volume, density, proportion with progressive motility, and proportion with normal morphology); to various derivatives from combinations of these variables; to seminal findings after vital staining; and to the PCT results. The best seminal predictor of fertility was the motile normal sperm density (MNSD), the 18 month conception rates being 57.4% +/- 4.6 (SE) and 30.2% +/- 5.9 (ratio 1.9, P less than 0.001) above and below a derived threshold value of 4 x 10(6)/ml. The PCT led to rates of 55.6% +/- 4.3 and 14.9% +/- 5.1 (ratio 3.73, P less than 0.001) for positive and negative results, respectively. The PCT also gave rise to a significantly distinct intermediate poor-positive sub-group (conception rate 30.6% +/- 9.0). Seminal analysis (the MNSD) did not affect the conception rate associated with a positive PCT but helped to discriminate further with a negative PCT (conception rates 22.5% +/- 8.7 with an MNSD above 4 x 10(6)/ml versus 5.6% +/- 4.8 below, P less than 0.05). The PCT was the single best predictor of fertility but seminal analysis (the MNSD) was of additional value after a negative PCT.

Specialist infertility practice was studied in a group of 708 couples within a population of residents of a single health district in England. They represented an annual incidence of 1.2 couples for every 1000 of the population. At least one in six couples needed specialist help at some time in their lives because of an average of infertility of 21/2 years, 71% of whom were trying for their first baby. Those attending gynaecology clinics made up 10% of new and 22% of all attendances. Failure of ovulation (amenorrhoea or oligomenorrhoea) occurred in 21% of cases and was successfully treated (two year conception rates of 96% and 78%). Tubal damage (14%) had a poor outlook (19%) despite surgery. Endometriosis accounted for infertility in 6%, although seldom because of tubal damage, cervical mucus defects or dysfunction in 3%, and coital failure in up to 6%. Sperm defects or dysfunction were the commonest defined cause of infertility (24%) and led to a poor chance of pregnancy (0-27%) without donor insemination. Obstructive azoospermia or primary spermatogenic failure was uncommon (2%) and hormonal causes of male infertility rare. Infertility was unexplained in 28% and the chance of pregnancy (overall 72%) was mainly determined by duration of infertility. In vitro fertilisation could benefit 80% of cases of tubal damage and 25% of unexplained infertility--that is, 18% of all cases, representing up to 216 new cases each year per million of the total population.