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Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways 1,2 . Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with βarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.

2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity., Competing Interests: Competing Interests: M.S. and L.L. are scientific co-founders of Angarus Therapeutics, which has exclusive licensing rights to patent PCT/US2018/50018. J.A.C., V.B., K.E.S., M.S., and L.L. are inventors on patent PCT/US2018/50018.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Fuzhenghuayu (FZHY) is a compound extracted from natural plants. Its anti-fibrotic effect has been confirmed in experimental and clinical studies. However, precise effects and underlying mechanisms of FZHY in liver angiogenesis largely remain understood. In this study, we investigated the effects of FZHY on sinusoidal capillarization and angiogenesis with mice challenged for Carbon tetrachloride (CCl 4 ) and dimethylnitrosamine (DMN), in vitro human hepatic sinusoidal endothelial cells (HHSEC) and Human Umbilical Vein Endothelial Cell (HUVEC) 3D fibrin gel model. Besides its anti-fibrotic effect, FZHY ameliorated CCl 4 and DMN-induced sinusoidal capillarization, angiogenesis and expression of angiogenesis-associated factors, i.e. CD31, VEGF, VEGF receptor II, phosphor-ERK and HIF-1α. Consistent with the findings based on animal models, inhibitory effects of FZHY on capillarization and angiogenesis were further confirmed in HHSEC and the HUVEC 3D fibrin gel model, respectively. These data suggest that FZHY ameliorates not only liver fibrosis but also vessel remodeling in experimental models. Therefore, FZHY might be a potentially useful drug to treat liver cirrhosis in clinical practice.

Chronic hepatitis D (CHD) results from an infection with the hepatitis B virus and hepatitis D virus (HDV). CHD is the most severe form of human viral hepatitis. Current treatment options consist of interferon alfa, which is effective only in a minority of patients. Study of HDV molecular virology has resulted in new approaches entering clinical trials, with phase-3 studies the most advanced. These include the entry inhibitor bulevirtide, the nucleic acid polymer REP 2139-Ca, the farnesyltransferase inhibitor lonafarnib, and pegylated interferon lambda. This article summarizes the available data on these emerging therapeutics., (Published by Elsevier Inc.)

Prolonged exposure of CD8 + T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8 + T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα + CD8 + T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα + cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8 + T cell-killing in vivo. SIRPα + CD8 + T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8 + T cells during chronic infection expands the cytotoxic subset of SIRPα + CD8 + T cells.

Hepatitis delta virus (HDV) infection of humans was first reported in 1977, and now it is now estimated that 15-20 million people are infected worldwide. Infection with HDV can be an acute or chronic process that occurs only in patients with an hepatitis B virus infection. Chronic HDV infection commonly results in the most rapidly progressive form of viral hepatitis; it is the chronic viral infection that is most likely to lead to cirrhosis, and it is associated with an increased risk of hepatocellular carcinoma. HDV infection is the only chronic human hepatitis virus infection without a therapy approved by the US Food and Drug Administration. Peginterferon alfa is the only recommended therapy, but it produces unsatisfactory results. We review therapeutic agents in development, designed to disrupt the HDV life cycle, that might benefit patients with this devastating disease., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Although treatment of hepatitis C virus (HCV) infection has improved, the prevalence of alcoholic liver disease (ALD) has been increasing, so we need an updated estimate of the burden and etiology-specific mortality of chronic liver diseases. We studied trends in age-standardized mortality of chronic liver diseases in adults at least 20 years old in the United States from 2007 through 2016., Methods: We collected data from the US Census and National Center for Health Statistics mortality records and identified individuals with HCV infection, ALD, nonalcoholic fatty liver disease, or hepatitis B virus infection using ICD-10 codes. We obtained temporal mortality rate patterns using joinpoint trend analysis with estimates of annual percentage change (APC)., Results: Age-standardized HCV-related mortality increased from 7.17 per 100,000 persons in 2007 to 8.14 per 100,000 persons in 2013, followed by a marked decrease in the time period at which patients began receiving treatment with direct-acting antiviral agents (from 8.09 per 100,000 persons in 2014 to 7.15 per 100,000 persons in 2016). The APC in HCV mortality increased 2.0%/year from 2007 through 2014 but decreased 6.4%/year from 2014 through 2016. In contrast, age-standardized mortality increased for ALD (APC 2.3% from 2007 through 2013 and APC 5.5% from 2013 through 2016) and nonalcoholic fatty liver disease (APC 6.1% from 2007 through 2013 and APC 11.3% from 2013 through 2016). Mortality related to hepatitis B virus decreased steadily from 2007 through 2016, with an average APC of -2.1% (95% CI -3.0 to -1.2). Etiology-based mortality in minority populations was higher. HCV-related mortality (per 100,000 persons) was highest in non-Hispanic blacks (10.28) and whites (6.92), followed by Hispanics (5.94), and lowest in non-Hispanic Asians (2.33). Non-Hispanic Asians had higher mortality for hepatitis B virus infection (2.82 per 100,000 vs 1.02 for non-Hispanic blacks and 0.47 for non-Hispanic whites)., Conclusion: In our population-based analysis of chronic liver disease mortality in the United States, the decrease in HCV-related mortality coincided with the introduction of direct-acting antiviral therapies, whereas mortality from ALD and nonalcoholic fatty liver disease increased during the same period. Minorities in the United States have disproportionately higher mortality related to chronic liver disease., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In this study, a real-time reverse transcription-polymerase chain reaction (real time RT-PCR) assay targeting 2 genetic segments was established to detect HDV RNA. Utilizing the World Health Organization International Standard for Hepatitis D Virus RNA, the lower limit of detection was 575 IU/mL, and the linearity of quantification ranged from 575,000 IU/mL to 575 IU/mL. 384 HBsAg-positive samples collected from China were tested by this method and HDV antibody detection. Eleven samples were positive for anti-HDV IgG which may persist after HDV resolution, 6 samples were HDV RNA positive, and 5 samples were positive for anti-HDV IgM. This assay showed more sensitivity than the detection of anti-HDV IgM. These data demonstrate that the real-time RT-PCR assay for HDV RNA could be implemented in the clinical detection of HDV infection in chronic HBV-infected patients in China., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hepatitis delta virus (HDV) causes the most severe form of human viral hepatitis. HDV requires a hepatitis B virus (HBV) coinfection to provide HDV with HBV surface antigen envelope proteins. The net effect of HDV is to make the underlying HBV disease worse, including higher rates of hepatocellular carcinoma. Accurate assessments of current HDV prevalence have been hampered by the lack of readily available and reliable quantitative assays, combined with the absence of a Food and Drug Administration-approved therapy. We sought to develop a convenient assay for accurately screening populations and to use this assay to determine HDV prevalence in a population with abnormally high rates of hepatocellular carcinoma. We developed a high-throughput quantitative microarray antibody capture assay for anti-HDV immunoglobulin G wherein recombinant HDV delta antigen is printed by microarray on slides coated with a noncontinuous, nanostructured plasmonic gold film, enabling quantitative fluorescent detection of anti-HDV antibody in small aliquots of patient serum. This assay was then used to screen all HBV-infected patients identified in a large randomly selected cohort designed to represent the Mongolian population. We identified two quantitative thresholds of captured antibody that were 100% predictive of the sample either being positive on standard western blot or harboring HDV RNA detectable by real-time quantitative PCR. Subsequent screening of the HBV + cohort revealed that a remarkable 57% were RNA + and an additional 4% were positive on western blot alone., Conclusion: The quantitative microarray antibody capture assay's unique performance characteristics make it ideal for population screening; its application to the Mongolian HBV surface antigen-positive population reveals an apparent ∼60% prevalence of HDV coinfection among these HBV-infected Mongolian subjects, which may help explain the extraordinarily high rate of hepatocellular carcinoma in Mongolia. (Hepatology 2017;66:1739-1749)., (© 2016 by the American Association for the Study of Liver Diseases.)

In humans, hepatitis D virus (HDV) infection only occurs in the presence of a concomitant hepatitis B virus (HBV) infection, and induces the most severe form of human viral hepatitis. Even though HDV is spread worldwide and is endemic in some regions, screening and treatment has been often neglected in part due to the lack of an effective therapy. Moreover, HDV prevalence rates are increasing in many countries driven by immigration from areas of high endemicity. Currently, no FDA-approved anti-HDV therapy is available, although interferon (IFN) alpha therapy has demonstrated benefit in a minority of patients. In this review, we present a current view of our understanding of the epidemiology, molecular virology and management of HDV infection. We additionally discuss new treatment approaches in development and describe the most promising results of recent and ongoing clinical trials of these new potential agents., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although currently available therapies for chronic hepatitis B virus infection can suppress viremia and provide long-term benefits for patients, they do not lead to a functional cure for most patients. Advances in our understanding of the virus-host interaction and the recent remarkable success of immunotherapy in cancer offer new and promising strategies for developing immune modulators that may become important components of a total therapeutic approach to hepatitis B, some of which are now in clinical development. Among the immunomodulatory agents currently being investigated to combat chronic HBV are toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, and engineered T cells. The efficacy of some immune modulatory therapies is compromised by high viral antigen levels. Cutting edge strategies, including RNA interference and CRISPR/Cas9, are now being studied that may ultimately be shown to have the capacity to lower viral antigen levels sufficiently to substantially increase the efficacy of these agents. The current advances in therapies for chronic hepatitis B are leading us toward the possibility of a functional cure., Competing Interests: Edward A. Pham, Dr. Ryan B. Perumpail, Benjamin J. Fram, Dr. Jeffrey S. Glenn, and Dr. Aijaz Ahmed report that they have no conflicts of interest. Dr. Robert G. Gish reports grants/research support from Gilead, Merck, Benitec, AbbVie; consultancy/advising for Abbott, AbbVie, Akshaya, Alexion, Arrowhead, Astra-Zeneca, Bayer AG, Bristol-Myers Squibb Company, Contravir, Eiger, Enyo, Genentech, Gilead Sciences, Hoffmann-LaRoche Ltd., Humabs, Intellia, Intercept, Ionis Pharmaceuticals, Isis, Janssen, MedImmune, MERCK, Nanogen, Novira; and Quest; speaker’s contracts with Alexion, Bayer, BMS, Gilead Sciences Inc., Salix/Valeant, AbbVie, and Merck; and stock in Kinex, Synageva, RiboSciences, CoCrystal, and Arrowhead; outside the submitted work. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Therapy for hepatitis C virus (HCV) infection is on the cusp of a new era. Until now, standard-of-care therapy has involved interferon (IFN) and ribavirin. With the first successful Phase III trials of specific targeted antiviral therapy for HCV (STAT-C) compounds, as well as three trials in progress giving the first glimpse of IFN-free combinations of STAT-C agents, this review looks ahead to the new classes of anti-HCV agents currently in clinical development. Successful pharmacologic control of HIV and TB frames the discussion, as well as consideration of the mutation frequency of HCV replication. Maximizing synergy between agents and minimizing cumulative toxicity will be critical to the design of future IFN-free STAT-C regimens., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cell encapsulating poly(ethylene glycol) hydrogels represent a promising approach for constructing 3D cultures designed to more closely approximate in vivo tissue environment. Improved strategies are needed, however, to optimally balance hydrogel permeability to support metabolic activities of encapsulated cells, while maintaining patternability to restore key aspects of tissue architecture. Herein, we have developed one such strategy incorporating hydrophobic nanoparticles to partially induce looser cross-linking density at the particle-hydrogel interface. Strikingly, our network design significantly increased hydrogel permeability, while only minimally affecting the matrix mechanical strength or prepolymer viscosity. This structural advantage improved viability and functions of encapsulated cells and permitted micron-scale structures to control over spatial distribution of incorporated cells. We expect that this design strategy holds promise for the development of more advanced artificial tissues that can promote high levels of cell metabolic activity and recapitulate key architectural features.

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

The N-terminal region of the hepatitis C virus (HCV) nonstructural protein NS5A contains an amphipathic alpha-helix that is necessary and sufficient for NS5A membrane association. A synthetic peptide (AH) comprising this amphipathic helix is able to lyse lipid vesicles that serve as a model system for virus particles. Based on quartz crystal microbalance-dissipation (QCM-D) experiments, the degree of vesicle rupturing was found to be inversely related to vesicle size, with maximal activity in the size range of several medically important viruses. In order to confirm and further study vesicle rupture, dynamic light scattering (DLS) and atomic force microscopy (AFM) experiments were also performed. The size dependence of vesicle rupturing helps explain the peptide's observed effect on the infectivity of a wide range of viruses. Further, in vitro studies demonstrated that AH peptide treatment significantly decreased the infectivity of HCV particles. Thus, the AH peptide might be used to rupture HCV particles extra-corporally (for HCV prevention) and within infected individuals (for HCV therapy).

Background & Aims: New therapies are needed to treat patients infected with hepatitis C virus (HCV), a major worldwide cause of chronic liver disease. Nitazoxanide (NTZ), originally used to treat cryptosporidiosis infection, recently was shown to have unexpected antiviral activity in the HCV replicon system and in chronically infected patients. A pilot clinical study suggested that NTZ can augment the antiviral effect of interferon (IFN), although the molecular basis for its effect was unknown., Methods: We analyzed the effects of NTZ on the regulation of eukaryotic initiation factor-2alpha (eIF2alpha) and its IFN-induced kinase, protein kinase activated by double-stranded RNA (PKR), in cells that support HCV RNA replication and in vitro biochemical assays., Results: NTZ increased eIF2alpha phosphorylation, a modification known to mediate host cell antiviral defenses. The addition of IFN to cell cultures increased NTZ-induced eIF2alpha phosphorylation. NTZ also increased PKR phosphorylation. In vitro, NTZ promoted PKR autophosphorylation, a key step in activating PKR's kinase activity for eIF2alpha. Finally, NTZ-induced eIF2alpha phosphorylation was reduced in the presence of specific inhibitors of PKR autophosphorylation., Conclusions: An important mechanism of NTZ's action involves activation of PKR, a key kinase that regulates the cell's innate antiviral response. These observations could explain the clinical antiviral effect of NTZ. NTZ might represent a new class of small molecules capable of potentiating and recapitulating important antiviral effects of IFN.

We have used simultaneous quartz crystal microbalance-dissipation (QCM-D) monitoring and four-detector optical reflectometry to monitor in situ the structural transformation of intact vesicles to a lipid bilayer on a gold surface. The structural transformation of lipid vesicles to a bilayer was achieved by introducing a particular amphipathic, alpha-helical (AH) peptide. The combined experimental apparatus allows us to simultaneously follow the acoustic and optical property changes of the vesicle rupturing process upon interaction with AH peptides. While QCM-D and reflectometry have similar sensitivities in terms of mass and thickness resolution, there are unique advantages in operating these techniques simultaneously on the same substrate. These advantages permit us to (1) follow the complex interaction between AH peptides and intact vesicles with both acoustic and optical mass measurements, (2) calculate the amount of dynamically coupled water during the interaction between AH peptides and intact vesicles, (3) demonstrate that the unexpectedly large increase of both adsorbed mass and the film's energy dissipation is mainly caused by swelling of the vesicles during the binding interaction with AH peptides, and (4) permit us to understand the structural transformation from intact vesicles to a bilayer via the AH peptide interaction by monitoring viscoelastic properties, acoustic mass, optical mass, and thickness changes of both the binding and destabilization processes. From the deduced "hydration signature" we followed the complex transformation of lipid assemblies. On the basis of this information, a mechanism of this structural transformation is proposed that provides new insight into the process of vesicle fusion on solid substrates.

HCV infection is an important cause of liver disease worldwide-nearly 80% of infected patients develop chronic liver disease, which leads to the development of liver cirrhosis and hepatocellular carcinoma. The ability of HCV to persist within a host is believed to be related to the numerous mechanisms by which it evades the immune response of the host. These mechanisms can be divided into defensive and offensive strategies. Examples of defensive mechanisms include replication within enclosed structures, which provides protection from the host's antiviral defenses, genetic diversity created by inaccurate replication, which yields mutants resistant to the cell's antiviral strategies, and association of the virion with protective lipoproteins. Offensive mechanisms include virally encoded proteins and other factors that disrupt the ability of the host cells to detect the virus and downregulate its ability to respond to interferon, impair innate immune defense mechanisms and alter T-cell responses, and prevent the development of an effective B-cell-mediated humoral response. Greater understanding of these viral survival strategies will ultimately translate into more effective antiviral therapies and better prognosis for patients.

Research on basic cellular processes involving local production or delivery of proteins, such as activity-dependent synaptic modification in neurons, would benefit greatly from a robust, nontoxic method to visualize selectively newly synthesized copies of proteins of interest within cells, tissues, or animals. We report a technique for covalent labeling of newly synthesized proteins of interest based on drug-dependent preservation of epitope tags. Epitope tags are removed from proteins of interest immediately after translation by the activity of a sequence-specific protease until the time a protease inhibitor is added, after which newly synthesized protein copies retain their tags. This method, which we call TimeSTAMP for time-specific tagging for the age measurement of proteins, allows sensitive and nonperturbative visualization and quantification of newly synthesized proteins of interest with exceptionally tight temporal control. We demonstrate applications of TimeSTAMP in retrospectively identifying growing synapses in cultured neurons and in visualizing the distribution of recently synthesized proteins in intact fly brains.

The hepatitis delta virus (HDV) is a defective RNA virus that requires the help of hepatitis B virus (HBV) for virion assembly and penetration into hepatocytes. Thus, it can only cause disease in individuals who carry HBV. HDV infection is present worldwide, predominantly in tropical and subtropical areas. However, with increased HBV control, HDV infection has significantly declined, although stably persisting, in developed countries. Most HDV-infected individuals develop chronic hepatitis D, and in approximately 80% of those cases, chronic hepatitis D progresses to cirrhosis within 5 to 10 years. The only approved therapy for chronic hepatitis D is interferon-alpha. Although transplantation offers a safe therapeutic option for managing end-stage HDV disease, novel therapeutic approaches are urgently needed.

By analyzing the viscoelastic properties of two distinct layers, a layer of "soft" vesicles and a "rigid" bilayer, we have created a model system to permit the study of film behavior in the region of nonlinear mass and frequency change (non-Sauerbrey). The structural transformation of lipid vesicles to a bilayer is shown to be accompanied by significant changes in their physical properties. After the adsorption and saturation of intact vesicles on gold surfaces, the adsorbed vesicle layer exhibits a soft, water-rich, viscoelastic state. The AH peptide, a vesicle-destabilizing agent, is then added to trigger the formation of a much thinner (approximately 5 nm), compact, and rigid bilayer. In this study, we used the quartz crystal microbalance with dissipation technique. Large non-Sauerbrey frequency and energy dissipation changes characterize the viscoelastic nature of adsorbed intact vesicle films thicker than approximately 10 nm. Once the transformation is complete, the frequency changes along with zero energy dissipation for sufficiently thin films (t approximately 5 nm) were effectively modeled with the Sauerbrey equation. Furthermore, we checked the validity of the Voigt-Voinova model in which the quartz substrate is treated as a Voigt element, which is beyond the Sauerbrey description. The calculations treating the film as having a constant viscosity agreed well with the Voigt-Voinova model. These results were compared to calculations done using the electromechanical (EM) model, which does not require a series expansion. The Voigt-Voinova results were in excellent agreement with the EM model, providing evidence that the expansion used in their study is quite accurate.