Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.</p> <p>Results</p> <p>A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg⁺⁺- and Mn⁺⁺-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn⁺⁺than Mg⁺⁺. Although the pH optima for these two enzymes are similar with Mn⁺⁺, they differ significantly in the presence of Mg⁺⁺, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn⁺⁺.</p> <p>Conclusion</p> <p>A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.</p

Etiology of pulmonary hypertension in multiple myeloma: A case series and literature review

BackgroundMultiple myeloma is often complicated by pulmonary hypertension through a variety of mechanisms. These mechanisms include pulmonary hypertension (PH) due to concomitant cardiac amyloid, high output heart failure due to anemia or lytic bone lesions, chronic thromboembolic pulmonary hypertension (CTEPH), toxicity from medications to treat multiple myeloma, and congestive heart failure. This case series highlights the various mechanisms through which multiple myeloma patients develop pulmonary hypertension.ObjectivesTo identify the etiologies of pulmonary hypertension and their management among multiple myeloma patients treated at University of California San Diego.MethodsA retrospective chart review was performed to identify patients with multiple myeloma and pulmonary hypertension who were evaluated at the University of California San Diego between July 2013 and July 2021. Patients also required a right heart catheterization to be included. Demographics, comorbidities, clinical course, and etiology of pulmonary hypertension were obtained from chart review.ResultsThere were 11 patients included. Of the 11 patients described, two had PH due to cardiac amyloid, one had PH due to high output heart failure, one had PH due to CTEPH, two had pulmonary arterial hypertension due to medications (carfilzomib), and five had PH due to congestive heart failure. The right heart catheterization and echocardiogram findings of the various mechanisms of PH in multiple myeloma are described.ConclusionsPulmonary hypertension in multiple myeloma is a common finding that necessitates further evaluation. The initial evaluation should include an echocardiogram and thorough medication review. Further diagnostic testing should be guided by the patient's history and can include right heart catheterization, cardiac biopsy, ventilation-perfusion scan, and bone scan

Molecular mechanisms of anthracycline cardiovascular toxicity

Anthracyclines are effective chemotherapeutic agents, commonly used in the treatment of a variety of hematologic malignancies and solid tumors. However, their use is associated with a significant risk of cardiovascular toxicities and may result in cardiomyopathy and heart failure. Cardiomyocyte toxicity occurs via multiple molecular mechanisms, including topoisomerase II-mediated DNA double-strand breaks and reactive oxygen species (ROS) formation via effects on the mitochondrial electron transport chain, NADPH oxidases (NOXs), and nitric oxide synthases (NOSs). Excess ROS may cause mitochondrial dysfunction, endoplasmic reticulum stress, calcium release, and DNA damage, which may result in cardiomyocyte dysfunction or cell death. These pathophysiologic mechanisms cause tissue-level manifestations, including characteristic histopathologic changes (myocyte vacuolization, myofibrillar loss, and cell death), atrophy and fibrosis, and organ-level manifestations including cardiac contractile dysfunction and vascular dysfunction. In addition, these mechanisms are relevant to current and emerging strategies to diagnose, prevent, and treat anthracycline-induced cardiomyopathy. This review details the established and emerging data regarding the molecular mechanisms of anthracycline-induced cardiovascular toxicity

Carfilzomib‐associated pulmonary arterial hypertension in multiple myeloma

Drug-induced pulmonary arterial hypertension (PAH) is constantly evolving as new drugs are developed. Carfilzomib is a recently approved therapy for relapsed and refractory multiple myeloma. While it has been associated with cardiovascular adverse events, such as ischemic heart disease and heart failure, PAH has not been a well-described side effect. We present two patients who developed PAH associated with initiation of carfilzomib. They both initially presented with severe dyspnea, had elevated right ventricular systolic pressure on transthoracic echocardiography and ultimately underwent right heart catheterization. With discontinuation of carfilzomib, both patients had improvement in hemodynamics. However, one patient required initiation of PAH-targeted therapies and has had worsening right ventricular function again despite permanent discontinuation of carfilzomib. It is important to recognize the association between carfilzomib and PAH. Echocardiography can be an important initial screening tool. PAH from carfilzomib therapy may be reversible, especially if diagnosed early; however, extended follow-up is essential

Genome-Wide Analyses of Avian Sarcoma Virus Integration Sites

The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design

High-Frequency Epigenetic Repression and Silencing of Retroviruses Can Be Antagonized by Histone Deacetylase Inhibitors and Transcriptional Activators, but Uniform Reactivation in Cell Clones Is Restricted by Additional Mechanisms

Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(−)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(−) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(−) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(−) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(−) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms

Automated cardiac volume assessment and cardiac long- and short-axis imaging plane prediction from electrocardiogram-gated computed tomography volumes enabled by deep learning.

AimsTo develop an automated method for bloodpool segmentation and imaging plane re-slicing of cardiac computed tomography (CT) via deep learning (DL) for clinical use in coronary artery disease (CAD) wall motion assessment and reproducible longitudinal imaging.Methods and resultsOne hundred patients who underwent clinically indicated cardiac CT scans with manually segmented left ventricle (LV) and left atrial (LA) chambers were used for training. For each patient, long-axis (LAX) and short-axis planes were manually defined by an imaging expert. A DL model was trained to predict bloodpool segmentations and imaging planes. Deep learning bloodpool segmentations showed close agreement with manual LV [median Dice: 0.91, Hausdorff distance (HD): 6.18 mm] and LA (Dice: 0.93, HD: 7.35 mm) segmentations and a strong correlation with manual ejection fraction (Pearson r: 0.95 LV, 0.92 LA). Predicted planes had low median location (6.96 mm) and angular orientation (7.96 ° ) errors which were comparable to inter-reader differences (P &gt; 0.71). 84-97% of DL-prescribed LAX planes correctly intersected American Heart Association segments, which was comparable (P &gt; 0.05) to manual slicing. In a test cohort of 144 patients, we evaluated the ability of the DL approach to provide diagnostic imaging planes. Visual scoring by two blinded experts determined ≥94% of DL-predicted planes to be diagnostically adequate. Further, DL-enabled visualization of LV wall motion abnormalities due to CAD and provided reproducible planes upon repeat imaging.ConclusionA volumetric, DL approach provides multiple chamber segmentations and can re-slice the imaging volume along standardized cardiac imaging planes for reproducible wall motion abnormality and functional assessment

Automated cardiac volume assessment and cardiac long- and short-axis imaging plane prediction from electrocardiogram-gated computed tomography volumes enabled by deep learning.

AimsTo develop an automated method for bloodpool segmentation and imaging plane re-slicing of cardiac computed tomography (CT) via deep learning (DL) for clinical use in coronary artery disease (CAD) wall motion assessment and reproducible longitudinal imaging.Methods and resultsOne hundred patients who underwent clinically indicated cardiac CT scans with manually segmented left ventricle (LV) and left atrial (LA) chambers were used for training. For each patient, long-axis (LAX) and short-axis planes were manually defined by an imaging expert. A DL model was trained to predict bloodpool segmentations and imaging planes. Deep learning bloodpool segmentations showed close agreement with manual LV [median Dice: 0.91, Hausdorff distance (HD): 6.18 mm] and LA (Dice: 0.93, HD: 7.35 mm) segmentations and a strong correlation with manual ejection fraction (Pearson r: 0.95 LV, 0.92 LA). Predicted planes had low median location (6.96 mm) and angular orientation (7.96 ° ) errors which were comparable to inter-reader differences (P &gt; 0.71). 84-97% of DL-prescribed LAX planes correctly intersected American Heart Association segments, which was comparable (P &gt; 0.05) to manual slicing. In a test cohort of 144 patients, we evaluated the ability of the DL approach to provide diagnostic imaging planes. Visual scoring by two blinded experts determined ≥94% of DL-predicted planes to be diagnostically adequate. Further, DL-enabled visualization of LV wall motion abnormalities due to CAD and provided reproducible planes upon repeat imaging.ConclusionA volumetric, DL approach provides multiple chamber segmentations and can re-slice the imaging volume along standardized cardiac imaging planes for reproducible wall motion abnormality and functional assessment

Inhibition of Coxsackievirus-associated dystrophin cleavage prevents cardiomyopathy

Heart failure in children and adults is often the consequence of myocarditis associated with Coxsackievirus (CV) infection. Upon CV infection, enteroviral protease 2A cleaves a small number of host proteins including dystrophin, which links actin filaments to the plasma membrane of muscle fiber cells (sarcolemma). It is unknown whether protease 2A–mediated cleavage of dystrophin and subsequent disruption of the sarcolemma play a role in CV-mediated myocarditis. We generated knockin mice harboring a mutation at the protease 2A cleavage site of the dystrophin gene, which prevents dystrophin cleavage following CV infection. Compared with wild-type mice, we found that mice expressing cleavage-resistant dystrophin had a decrease in sarcolemmal disruption and cardiac virus titer following CV infection. In addition, cleavage-resistant dystrophin inhibited the cardiomyopathy induced by cardiomyocyte-restricted expression of the CV protease 2A transgene. These findings indicate that protease 2A–mediated cleavage of dystrophin is critical for viral propagation, enteroviral-mediated cytopathic effects, and the development of cardiomyopathy