H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z[superscript AP3]) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z[superscript AP3] interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z[superscript AP3] was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z[superscript AP3] ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z[superscript AP3] ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z[superscript AP3] displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z[superscript AP3] mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.Massachusetts Life Sciences Center (David H. Koch Institute for Integrative Cancer Research at MIT Core Grant P30-CA14051)National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (Grant CBET-0939511)MIT Faculty Start-up FundMassachusetts Institute of Technology. Computational and Systems Biology Initiative (Merck & Co. Postdoctoral Fellowship

National and subnational estimation of the prevalence of peripheral artery disease (PAD) in China:a systematic review and meta-analysis

Background: Peripheral artery disease (PAD), the third leading cause of atherosclerotic vascular morbidity, affects approximately 202 million people worldwide, among whom more than two-thirds reside in low- and middle-income countries (LMIC). For China, the largest developing country, little is known about the epidemiology of PAD. We aimed to estimate the prevalence of PAD and the number of affected people in China, establish the main risk factors for PAD and assess the number of people with PAD at the sub-national level.Methods: We searched China National Knowledge Infrastructure (CNKI), Wanfang, Chinese Biomedicine Literature Database (CBM-SinoMed), PubMed, Embase and Medline for population-based studies that have reported the prevalence of PAD in the general Chinese population from 1990 onwards. PAD was defined as an ankle-brachial index (ABI) lower than or equal to 0.90. We used a multilevel mixed-effects logistic regression to generate the gender- and age-specific prevalence of PAD, and a random-effects meta-analysis to pool the odds ratios (ORs) of major risk factors. United Nations population numbers were used to estimate and project the number of affected people from 2000 to 2020. Finally, we used the risk factors-based model to distribute the national number of people with PAD into different settings (urban and rural) and regions (East, Central and West) for the year 2010.Results: Overall, 37 articles met all inclusion criteria and provided prevalence estimates, among which 14 also explored risk factors for PAD. The prevalence of PAD increased gradually by age until mid-60s, after which the increase accelerated. In males, the prevalence of PAD ranged from 2.81% (95% CI = 1.77-4.43) in those aged 25-29 years to 21.95% (95% CI = 15.39-30.31) in those 95-99 years old. In females, the PAD prevalence increased from 3.84% (95% CI = 2.44-5.98) in those aged 25-29 years to 27.95% (95% CI = 20.14-37.37) in those aged 95-99 years. The PAD prevalence was consistently higher in females than in males across all age groups. This difference was most significantly pronounced among the elderly, starting from 60 years. Between 2000 and 2020, the total number of Chinese people with PAD is expected to increase by 40%: from 29.44 million (95% CI = 22.51-38.62) in 2000 to 41.13 million (95% CI = 32.00-52.95) in 2020. Current smoking was the strongest risk factor for PAD, with a meta-odds ratio (OR) of 2.62 (95% CI = 1.44-4.76), followed by hypertension (1.94, 95% CI = 1.48-2.53) and diabetes (1.71, 95% CI = 1.45-2.01). In 2010, 15.18 million (95% CI = 11.74-19.67) people with PAD resided in the East China, 11.08 million (95% CI = 8.61-14.28) in the Central China and 8.65 million (95% CI = 6.71-11.16) in the West China. In addition, 24.20 million (95% CI = 18.82-31.16) people with PAD were living in rural areas, accounting for almost 70% of all PAD cases in China.Conclusions: With rapid ageing in China, PAD has become a serious public health problem. More research and optimal interventions on PAD are required to better identify effective strategies for prevention and treatment of PAD in China.</p

Characterization of receptor use and entry mechanisms in two KSHV infection systems

Viruses initiate infection at the cell surface, where they use viral proteins to contact and manipulate naturally occurring host receptors in the plasma membrane. Through this interaction, viruses negotiate internalization and begin their infection cycle. These virus-receptor interactions can be surprisingly complex, sometimes coordinating many receptors using several viral proteins simultaneously. Cytoskeletal rearrangements, a multitude of intracellular signaling cascades, and even transcriptional changes can be triggered through the host receptors by this initial interaction and influence the outcome of the attempted infection. Thus, viral entry is a nuanced process evolved to ensure that viruses can infect the right cells at the right time, while successfully evading host defenses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an important human pathogen. It is the causative agent of several cancers and inflammatory disease which together, in the context of the global HIV epidemic, are a major public health burden. KSHV is the most recent of the human herpesviruses to be discovered, but research on KSHV entry mechanisms has almost a twenty-year history. Eight receptors for KSHV have been described, and it has become apparent that the step-by-step details of KSHV entry mechanisms are likely to be unique in every cell line. By interacting with the same set of receptors on human foreskin fibroblasts or primary microvascular endothelial cells, for example, the virion is internalized by clathrin-mediated endocytosis or clathrin-independent macropinocytosis, respectively. Here we investigated KSHV receptor usage in cell types that are relatively understudied in the field: epithelial cells and lymphocytes. We uncovered novel variability in receptor use across many susceptible cell lines, particularly that infection of epithelial cells and lymphocytes was independent of known KSHV integrin receptors and likely all known integrins. Additionally, we found that infection of Caki-1 and HeLa cells did not require EphA2 signaling, and infection of primary oral keratinocytes did not depend on Eph receptor interactions whatsoever. We hypothesize that there is at least one more KSHV receptor required for infection in the epithelial cells we studied. Furthermore, we showed that coculture-mediated infection of BJAB cells required heparan sulfate and Eph receptor interactions, despite the fact that BJAB cells do not express heparan sulfate and manipulation of Eph receptor expression did not affect infection. These results are evocative of a “transfer infection” mechanism akin to Epstein-Barr Virus, which requires receptor interactions on adjacent cells to promote infection of an otherwise non-susceptible cell type. We identified KSHV orf28 as a potential player in determining lymphocyte tropism.Our work reveals another layer of complexity beyond receptor availability on cells. It is now clear that even when KSHV receptors are expressed by a cell, additional contextual factors determine whether they play a role during infection. Going forward, this will be very important to understand, especially since virus-receptor interactions are often targeted by small molecules or biologics in the hopes of slowing viral dissemination

Characterization of receptor use and entry mechanisms in two KSHV infection systems

Viruses initiate infection at the cell surface, where they use viral proteins to contact and manipulate naturally occurring host receptors in the plasma membrane. Through this interaction, viruses negotiate internalization and begin their infection cycle. These virus-receptor interactions can be surprisingly complex, sometimes coordinating many receptors using several viral proteins simultaneously. Cytoskeletal rearrangements, a multitude of intracellular signaling cascades, and even transcriptional changes can be triggered through the host receptors by this initial interaction and influence the outcome of the attempted infection. Thus, viral entry is a nuanced process evolved to ensure that viruses can infect the right cells at the right time, while successfully evading host defenses. Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an important human pathogen. It is the causative agent of several cancers and inflammatory disease which together, in the context of the global HIV epidemic, are a major public health burden. KSHV is the most recent of the human herpesviruses to be discovered, but research on KSHV entry mechanisms has almost a twenty-year history. Eight receptors for KSHV have been described, and it has become apparent that the step-by-step details of KSHV entry mechanisms are likely to be unique in every cell line. By interacting with the same set of receptors on human foreskin fibroblasts or primary microvascular endothelial cells, for example, the virion is internalized by clathrin-mediated endocytosis or clathrin-independent macropinocytosis, respectively. Here we investigated KSHV receptor usage in cell types that are relatively understudied in the field: epithelial cells and lymphocytes. We uncovered novel variability in receptor use across many susceptible cell lines, particularly that infection of epithelial cells and lymphocytes was independent of known KSHV integrin receptors and likely all known integrins. Additionally, we found that infection of Caki-1 and HeLa cells did not require EphA2 signaling, and infection of primary oral keratinocytes did not depend on Eph receptor interactions whatsoever. We hypothesize that there is at least one more KSHV receptor required for infection in the epithelial cells we studied. Furthermore, we showed that coculture-mediated infection of BJAB cells required heparan sulfate and Eph receptor interactions, despite the fact that BJAB cells do not express heparan sulfate and manipulation of Eph receptor expression did not affect infection. These results are evocative of a “transfer infection” mechanism akin to Epstein-Barr Virus, which requires receptor interactions on adjacent cells to promote infection of an otherwise non-susceptible cell type. We identified KSHV orf28 as a potential player in determining lymphocyte tropism.Our work reveals another layer of complexity beyond receptor availability on cells. It is now clear that even when KSHV receptors are expressed by a cell, additional contextual factors determine whether they play a role during infection. Going forward, this will be very important to understand, especially since virus-receptor interactions are often targeted by small molecules or biologics in the hopes of slowing viral dissemination

A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3β1, αVβ3, and αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCE Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types

A Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism is Independent of Integrins α3β1, αVβ3, and αVβ5

ABSTRACTHost receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins α3β1, αVβ3, αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCEOur data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.</jats:sec

Pharmacoeconomic savings associated with alternate dosing strategy of nivolumab and ipilimumab combination therapy.

79 Background: Nivolumab (NIVO) plus ipilimumab (IPI) is a commonly used therapy for advanced melanoma. Original weight-based dosing of NIVO 1 mg/kg plus IPI 3 mg/kg (NIVO1+IPI3) was approved in 2017. Recently, the CHECKMATE 511 trial demonstrated improved tolerability of NIVO 3 mg/kg plus IPI 1 mg/kg (NIVO3 + IPI1) compared to the original dosing regimen without observed differences in efficacy.Objective: To determine the pharmacoeconomic implications of NIVO1+IPI3 and NIVO3+IPI1 with a dose banding strategy applied. Methods: Patients with advanced melanoma (N = 21) who received IPI and NIVO in combination between 4/2019 – 7/2019 were evaluated via a single-center retrospective chart review. A total of 118 checkpoint inhibitor doses (59 NIVO, 59 IPI) were analyzed.Weapplied a dose banding strategy to both NIVO1+IPI3 and NIVO3+IPI1 regimens to examine pharmaceutical expenditures using the two dosing regimens, whichincluded our organization 10% dose-vial rounding policy. Pharmaceutical expenditure using average wholesale price (AWP), which was $32.42 per mg of NIVO and$180.03 per mg of IPI, was calculated for each dosing strategy (NIVO1+IPI3 and NIVO3+IPI1). Results: The anticipated cost savings of patients receiving NIVO3+IPI1 combination therapy compared to NIVO1+IPI3, (both with dose rounding strategy applied), was 1,459,473 or >70,000 per patient, representing a 47.2% savings from the original NIVO1 +IPI3 regimen (Table). Conclusions: In addition to improved tolerability and comparable efficacy for NIVO3+IPI1 vs NIVO1+IPI3, our study shows that adoption of the NIVO3+IPI1 regimen results in significant savings in drug costs. Considerations should be made in future combination trials to compare NIVO3+IPI1 as a treatment arm to NIVO1+IPI3 to see if comparable clinical results can be obtained with less toxicity and decreased pharmaceutical spending. [Table: see text] </jats:p