Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

SnapShot: Cancer Immunotherapy with Oncolytic Viruses.

Oncolytic viruses (OVs) preferentially infect and kill cancer cells without harming normal cells. OVs can revert cancer-associated immune suppression and initiate clinically meaningful antitumor immune responses. OVs and their resultant immunological events can act at both primary and metastatic sites. Thus, OVs can be exploited for cancer gene therapies and immunotherapies alone or in combination with other interventions, including immune checkpoint blockade.

Genomic and functional determinants of host spectrum in Group B Streptococcus.

Group B Streptococcus (GBS) is a major human and animal pathogen that threatens public health and food security. Spill-over and spill-back between host species is possible due to adaptation and amplification of GBS in new niches but the evolutionary and functional mechanisms underpinning those phenomena are poorly known. Based on analysis of 1,254 curated genomes from all major GBS host species and six continents, we found that the global GBS population comprises host-generalist, host-adapted and host-restricted sublineages, which are found across host groups, preferentially within one host group, or exclusively within one host group, respectively, and show distinct levels of recombination. Strikingly, the association of GBS genomes with the three major host groups (humans, cattle, fish) is driven by a single accessory gene cluster per host, regardless of sublineage or the breadth of host spectrum. Moreover, those gene clusters are shared with other streptococcal species occupying the same niche and are functionally relevant for host tropism. Our findings demonstrate (1) the heterogeneity of genome plasticity within a bacterial species of public health importance, enabling the identification of high-risk clones; (2) the contribution of inter-species gene transmission to the evolution of GBS; and (3) the importance of considering the role of animal hosts, and the accessory gene pool associated with their microbiota, in the evolution of multi-host bacterial pathogens. Collectively, these phenomena may explain the adaptation and clonal expansion of GBS in animal reservoirs and the risk of spill-over and spill-back between animals and humans.

Suppression of PKR promotes network excitability and enhanced cognition by interferon-γ-mediated disinhibition.

The double-stranded RNA-activated protein kinase (PKR) was originally identified as a sensor of virus infection, but its function in the brain remains unknown. Here, we report that the lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability. In addition, loss of PKR increases the late phase of long-lasting synaptic potentiation (L-LTP) in hippocampal slices. These effects are caused by an interferon-γ (IFN-γ)-mediated selective reduction in GABAergic synaptic action. Together, our results reveal that PKR finely tunes the network activity that must be maintained while storing a given episode during learning. Because PKR activity is altered in several neurological disorders, this kinase presents a promising new target for the treatment of cognitive dysfunction. As a first step in this direction, we show that a selective PKR inhibitor replicates the Pkr(-/-) phenotype in WT mice, enhancing long-term memory storage and L-LTP.

Fluctuating hydrodynamics and the Rayleigh-Plateau instability.

The Rayleigh-Plateau instability occurs when surface tension makes a fluid column become unstable to small perturbations. At nanometer scales, thermal fluctuations are comparable to interfacial energy densities. Consequently, at these scales, thermal fluctuations play a significant role in the dynamics of the instability. These microscopic effects have previously been investigated numerically using particle-based simulations, such as molecular dynamics (MD), and stochastic partial differential equation-based hydrodynamic models, such as stochastic lubrication theory. In this paper, we present an incompressible fluctuating hydrodynamics model with a diffuse-interface formulation for binary fluid mixtures designed for the study of stochastic interfacial phenomena. An efficient numerical algorithm is outlined and validated in numerical simulations of stable equilibrium interfaces. We present results from simulations of the Rayleigh-Plateau instability for long cylinders pinching into droplets for Ohnesorge numbers of Oh = 0.5 and 5.0. Both stochastic and perturbed deterministic simulations are analyzed and ensemble results show significant differences in the temporal evolution of the minimum radius near pinching. Short cylinders, with lengths less than their circumference, were also investigated. As previously observed in MD simulations, we find that thermal fluctuations cause these to pinch in cases where a perturbed cylinder would be stable deterministically. Finally, we show that the fluctuating hydrodynamics model can be applied to study a broader range of surface tension-driven phenomena.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Greenhouse gas emissions associated with suboptimal asthma care in the UK: the SABINA healthCARe-Based envirONmental cost of treatment (CARBON) study.

BACKGROUND: Poorly controlled asthma is associated with increased morbidity and healthcare resource utilisation (HCRU). Therefore, to quantify the environmental impact of asthma care, this retrospective, cohort, healthCARe-Based envirONmental cost of treatment (CARBON) study estimated greenhouse gas (GHG) emissions in the UK associated with the management of well-controlled versus poorly controlled asthma. METHODS: Patients with current asthma (aged ≥12 years) registered with the Clinical Practice Research Datalink (2008‒2019) were included. GHG emissions, measured as carbon dioxide equivalent (CO2e), were estimated for asthma-related medication use, HCRU and exacerbations during follow-up of patients with asthma classified at baseline as well-controlled (<3 short-acting ß2-agonist (SABA) canisters/year and no exacerbations) or poorly controlled (≥3 SABA canisters/year or ≥1 exacerbation). Excess GHG emissions due to suboptimal asthma control included ≥3 SABA canister prescriptions/year, exacerbations and any general practitioner and outpatient visits within 10 days of hospitalisation or an emergency department visit. RESULTS: Of the 236 506 patients analysed, 47.3% had poorly controlled asthma at baseline. Scaled to the national level, the overall carbon footprint of asthma care in the UK was 750 540 tonnes CO2e/year, with poorly controlled asthma contributing excess GHG emissions of 303 874 tonnes CO2e/year, which is equivalent to emissions from >124 000 houses in the UK. Poorly controlled versus well-controlled asthma generated 3.1-fold higher overall and 8.1-fold higher excess per capita carbon footprint, largely SABA-induced, with smaller contributions from HCRU. CONCLUSIONS: These findings suggest that addressing the high burden of poorly controlled asthma, including curbing high SABA use and its associated risk of exacerbations, may significantly alleviate asthma care-related carbon emissions.

Perioperative arginine prevents metastases by accelerating natural killer cell recovery after surgery.

Profound natural killer (NK) cell suppression after cancer surgery is a main driver of metastases and recurrence, for which there is no clinically approved intervention available. Surgical stress is known to cause systemic postoperative changes that negatively modulate NK cell function including the expansion of surgery-induced myeloid-derived suppressor cells (Sx-MDSCs) and a marked reduction in arginine bioavailability. In this study, we determine that Sx-MDSCs regulate systemic arginine levels in the postoperative period and that restoring arginine imbalance after surgery by dietary intake alone was sufficient to significantly reduce surgery-induced metastases in our preclinical murine models. Importantly, the effects of perioperative arginine were dependent upon NK cells. Although perioperative arginine did not prevent immediate NK cell immunoparalysis after surgery, it did accelerate their return to preoperative cytotoxicity, interferon gamma secretion, and activating receptor expression. Finally, in a cohort of patients with colorectal cancer, postoperative arginine levels were shown to correlate with their Sx-MDSC levels. Therefore, this study lends further support for the use of perioperative arginine supplementation by improving NK cell recovery after surgery.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

Surface coverage dynamics for reversible dissociative adsorption on finite linear lattices.

Dissociative adsorption onto a surface introduces dynamic correlations between neighboring sites not found in non-dissociative absorption. We study surface coverage dynamics where reversible dissociative adsorption of dimers occurs on a finite linear lattice. We derive analytic expressions for the equilibrium surface coverage as a function of the number of reactive sites, N, and the ratio of the adsorption and desorption rates. Using these results, we characterize the finite size effect on the equilibrium surface coverage. For comparable N's, the finite size effect is significantly larger when N is even than when N is odd. Moreover, as N increases, the size effect decays more slowly in the even case than in the odd case. The finite-size effect becomes significant when adsorption and desorption rates are considerably different. These finite-size effects are related to the number of accessible configurations in a finite system where the odd-even dependence arises from the limited number of accessible configurations in the even case. We confirm our analytical results with kinetic Monte Carlo simulations. We also analyze the surface-diffusion case where adsorbed atoms can hop into neighboring sites. As expected, the odd-even dependence disappears because more configurations are accessible in the even case due to surface diffusion.

Extracellular Vesicles and Viruses: Two Intertwined Entities.

Viruses share many attributes in common with extracellular vesicles (EVs). The cellular machinery that is used for EV production, packaging of substrates and secretion is also commonly manipulated by viruses for replication, assembly and egress. Viruses can increase EV production or manipulate EVs to spread their own genetic material or proteins, while EVs can play a key role in regulating viral infections by transporting immunomodulatory molecules and viral antigens to initiate antiviral immune responses. Ultimately, the interactions between EVs and viruses are highly interconnected, which has led to interesting discoveries in their associated roles in the progression of different diseases, as well as the new promise of combinational therapeutics. In this review, we summarize the relationships between viruses and EVs and discuss major developments from the past five years in the engineering of virus-EV therapies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infectivity by Viral Load, S Gene Variants and Demographic Factors, and the Utility of Lateral Flow Devices to Prevent Transmission.

BACKGROUND: How severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity varies with viral load is incompletely understood. Whether rapid point-of-care antigen lateral flow devices (LFDs) detect most potential transmission sources despite imperfect clinical sensitivity is unknown. METHODS: We combined SARS-CoV-2 testing and contact tracing data from England between 1 September 2020 and 28 February 2021. We used multivariable logistic regression to investigate relationships between polymerase chain reaction (PCR)-confirmed infection in contacts of community-diagnosed cases and index case viral load, S gene target failure (proxy for B.1.1.7 infection), demographics, SARS-CoV-2 incidence, social deprivation, and contact event type. We used LFD performance to simulate the proportion of cases with a PCR-positive contact expected to be detected using 1 of 4 LFDs. RESULTS: In total, 231 498/2 474 066 (9%) contacts of 1 064 004 index cases tested PCR-positive. PCR-positive results in contacts independently increased with higher case viral loads (lower cycle threshold [Ct] values), for example, 11.7% (95% confidence interval [CI] 11.5-12.0%) at Ct = 15 and 4.5% (95% CI 4.4-4.6%) at Ct = 30. B.1.1.7 infection increased PCR-positive results by ~50%, (eg, 1.55-fold, 95% CI 1.49-1.61, at Ct = 20). PCR-positive results were most common in household contacts (at Ct = 20.1, 8.7% [95% CI 8.6-8.9%]), followed by household visitors (7.1% [95% CI 6.8-7.3%]), contacts at events/activities (5.2% [95% CI 4.9-5.4%]), work/education (4.6% [95% CI 4.4-4.8%]), and least common after outdoor contact (2.9% [95% CI 2.3-3.8%]). Contacts of children were the least likely to test positive, particularly following contact outdoors or at work/education. The most and least sensitive LFDs would detect 89.5% (95% CI 89.4-89.6%) and 83.0% (95% CI 82.8-83.1%) of cases with PCR-positive contacts, respectively. CONCLUSIONS: SARS-CoV-2 infectivity varies by case viral load, contact event type, and age. Those with high viral loads are the most infectious. B.1.1.7 increased transmission by ~50%. The best performing LFDs detect most infectious cases.

Comprehensive, integrated, and phased whole-genome analysis of the primary ENCODE cell line K562.

K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and indels (both corrected for CN in aneuploid regions), loss of heterozygosity, megabase-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs), including small and large-scale complex SVs and nonreference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT Taking aneuploidy into account, we reanalyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Participatory research with a rural Aboriginal Community Controlled Health Organisation: lessons learned using the CONSIDER statement.

CONTEXT: In Australia, Aboriginal Community Controlled Health Organisations (ACCHOs) are geographically proximal to where Aboriginal and Torres Strait Islander People reside and are valued for providing holistic and culturally safe primary health care. Partnering with ACCHOs in research is appropriate for redressing health inequities experienced by Aboriginal and Torres Strait Islander People, which includes a high burden of chronic disease. Historically, some approaches to Aboriginal and Torres Strait Islander health research have been unethical. Greater accountability in the research process, transparency in reporting, and use of culturally appropriate research methodologies are key recommendations to improving the ethical integrity of research. The need for strengthening the reporting of health research involving Aboriginal and Torres Strait Islander People and Indigenous peoples globally led to the development of the CONSolIDated critERia for strengthening the reporting of health research involving Indigenous peoples (CONSIDER statement), which is a synthesis of international ethical guidelines. This project report uses the CONSIDER statement to critically reflect on participatory research undertaken in partnership with an ACCHO in the rural context and to identify lessons of value for future research. ISSUE: By using the CONSIDER statement as a tool for critical reflection, it was identified that processes used to establish a research partnership with an ACCHO were key to setting the research agenda, including identifying ethical issues, the needs of local Aboriginal and Torres Strait Islander People, and expectations from the research. The participation of Aboriginal community members throughout the entire research process was not only methodologically important but was also ethically appropriate. Research activities in this project included opportunities for Aboriginal community members to directly share their perspectives and experiences and develop local solutions to issues affecting them. Outcomes included evidence to support future funding applications, community-derived priorities that assisted with government reporting, and locally identified methods for addressing chronic disease management. Key to this was building the research capacity and capability of local Aboriginal community members, which also reflected the ethical principles of reciprocity and equity. This also provided opportunities for non-Indigenous researchers to learn from local Aboriginal community members and develop skills in culturally appropriate research. LESSONS LEARNED: Using the CONSIDER statement was beneficial in enabling researchers to critically reflect on a participatory research project undertaken in partnership with a rural ACCHO. Researchers identified that participatory approaches can be used to generate research of relevance to local Aboriginal community members and their ACCHOs, and to support health service reporting, and future funding applications. Research timelines and activities needed to be flexible and adaptable, to allow for staff turnover and unforeseen events of cultural significance. Similarly, it is important for researchers to be receptive to change and open to learning. Although research partnerships are established on trust and mutual respect, it is recommended that greater formal provisions are required to protect the intellectual property of Aboriginal and Torres Strait Islander communities involved in research. These lessons are likely to be transferrable to other settings and are of value to researchers seeking to partner with ACCHOs in research.

Changes in plant species abundance alter the multifunctionality and functional space of heathland ecosystems.

Though it is well established that species composition affects ecosystem function, the way in which species combine to control overall ecosystem functioning is still debated. In experimental mesocosms, we planted three functionally distinct dry-heath species in varying proportions and measured multiple ecosystem properties related to nutrient cycling and carbon storage (hereafter functions). Overall ecosystem functioning was described as the main axes of variation in ecosystem functioning (functional space) and the proportion of ecosystem functions at high levels; for example, fast carbon and nutrient cycling (cluster-based multifunctionality). The first functional space axis, related to nitrogen availability, was driven by plant species abundance, particularly that of legumes, which strongly affected many individual functions. The second, related to total plant biomass and woodiness, was mostly driven by the abundance of dwarf shrubs. Similarly, cluster-based multifunctionality was related to the initial abundance of all species, but particularly the legume. Interactions between species also affected ecosystem multifunctionality, but these effects were smaller in magnitude. These results indicate that species interactions could play a secondary role to species abundance and identity in driving the overall ecosystem functioning of heathlands, but also that axes of variation in functional space are clearly linked to plant functional composition.

cGAS-STING and Cancer: Dichotomous Roles in Tumor Immunity and Development.

cGMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) sensing has emerged as a key regulator of innate immune responses to both exogenous and endogenous DNA. Recent studies reveal critical roles for this pathway in natural antitumor immunity across cancer types as well as in immune checkpoint blockade therapy. However, it is also clear that some tumors evade cGAS-STING-mediated immune responses, and immunomodulatory therapeutics are currently being explored to target this pathway. Finally, we also discuss recent observations that cGAS-STING-mediated inflammation may promote tumor initiation, growth, and metastasis in certain malignancies and how this may complicate the utility of this pathway in therapeutic development.

Adenosquamous carcinoma of the breast, an uncommon diagnosis with poor prognosis-Lessons learned from a 12-year analysis of the National Cancer Database.

We compared the clinicopathologic characteristics and prognosis of adenosquamous carcinoma (ASQ) of the breast with invasive ductal carcinoma (IDC), utilizing the National Cancer Database (NCDB) from 2004 to 2015. 1 932 688 female patients had invasive breast carcinoma; 1 421 250 had IDC (73.5%); and 453 had ASQ (0.0002%). When compared to IDC, ASQ patients were significantly (P < .05) older and had grade 1 tumors; negative lymph nodes; ER/PR/HER2-negative tumors; and worse 5-year overall survival (64.9% vs 74%, respectively). Our study, largest to date on ASQ, revealed an aggressive carcinoma with a significantly worse prognosis than IDC. "Personalized medicine" treatment approach for patients with this uncommon carcinoma is needed.

Haplotype-resolved and integrated genome analysis of the cancer cell line HepG2.

HepG2 is one of the most widely used human cancer cell lines in biomedical research and one of the main cell lines of ENCODE. Although the functional genomic and epigenomic characteristics of HepG2 are extensively studied, its genome sequence has never been comprehensively analyzed and higher order genomic structural features are largely unknown. The high degree of aneuploidy in HepG2 renders traditional genome variant analysis methods challenging and partially ineffective. Correct and complete interpretation of the extensive functional genomics data from HepG2 requires an understanding of the cell line's genome sequence and genome structure. Using a variety of sequencing and analysis methods, we identified a wide spectrum of genome characteristics in HepG2: copy numbers of chromosomal segments at high resolution, SNVs and Indels (corrected for aneuploidy), regions with loss of heterozygosity, phased haplotypes extending to entire chromosome arms, retrotransposon insertions and structural variants (SVs) including complex and somatic genomic rearrangements. A large number of SVs were phased, sequence assembled and experimentally validated. We re-analyzed published HepG2 datasets for allele-specific expression and DNA methylation and assembled an allele-specific CRISPR/Cas9 targeting map. We demonstrate how deeper insights into genomic regulatory complexity are gained by adopting a genome-integrated framework.