Colorimetric detection of Cr(VI) in water using tetramethyl benzidine (TMB) as an indicator.

Hexavalent chromium (CrVI) poses a serious risk to both human and environment health. Hence, a simple, robust, and efficient analytical method must be developed to monitor the presence of Cr(VI) in the environment. The current investigation concentrated on the colorimetric detection of Cr(VI) using TMB as indicator in the presence of H2O2. The study found that Cr(VI) reacts with H2O2 to generate hydroxyl radicals which oxidize TMB in a concentration dependent manner. Under optimized conditions, the method obtained a good linearity range (0.025-0.5 mg/L, r2 = 0.9944) with LOD and LOQ of 0.009 mg/L and 0.029 mg/L, respectively. The technique was further improved by the addition of EDTA in the sample preparation protocol to reduce the false positive result by the presence of ions like Cu2+, Fe3+, etc. The study recorded improved Cr(VI) recoveries (81.73-111.40 %) at different fortification levels (0.1-0.5 mg/L). Under optimized conditions, the EDTA added method obtained a good linear response (r2 = 0.9952) with a detection limit of 0.023 mg/L which is less than the prescribed limits by WHO (0.05 mg/L) and US EPA (0.1 mg/L) for drinking water. The developed analytical method is very simple without use of any nanomaterial and the results with natural water samples show that it has the potential for real-time detection of Cr(VI) in the environment.

Cr-Detector: A simple chemosensing system for onsite Cr (VI) detection in water.

Due to the increase in urbanization and industrialization, the load of toxicants in the environment is alarming. The most common toxicants, including heavy metals and metalloids such as hexavalent Chromium, have severe pathophysiological impacts on humans and other aquatic biotas. Therefore, developing a portable rapid detection device for such toxicants in the aquatic environment is necessary. This work portrays the development of a field-portable image analysis device coupled with 3,3',5,5'-tetramethylbenzidine (TMB) as a sensing probe for chromium (VI) detection in the aquatic ecosystem. Sensor parameters, such as reagent concentration, reaction time, etc., were optimized for the sensor development and validation using a commercial UV-Vis spectrophotometer. The chemoreceptor integrated with a uniform illumination imaging system (UIIS) revealed the system's applicability toward Cr(VI) detection. The calibration curve using the R-value of image parameters allows Cr(VI) detection in the linear range of 25 to 600 ppb, which covers the prescribed permissible limit by various regulatory authorities. Furthermore, the adjusted R2 = 0.992 of the linear fit and correlation coefficients of 0.99018 against the spectrophotometric method signifies the suitability of the developed system. This TMB-coupled field-portable sensing system is the first-ever reported image analysis-based technology for detecting a wide range of Cr(VI) in aquatic ecosystems to our knowledge.

Environmental bisphenol A disrupts methylation of steroidogenic genes in the ovary of Paradise threadfin Polynemus paradiseus via abnormal DNA methylation: Implications for human exposure and health risk assessment.

Bisphenol A, endocrine-disrupting chemicals (EDCs) impacting disease development via epigenetic modifications, is crucial in transcriptional regulation. However, ecotoxicology's limited exploration of epigenetics prompted our study's objective: examining the extended exposure of riverine Bisphenol A (BPA), a potent EDC, on DNA methylation during female paradise threadfin (Polynemus paradiseus) reproductive maturation. Assessing BPA contamination in riverine water, we collected fish samples from two locations with distinct contamination levels. In the highly contaminated region (Hc), we observed elevated DNA methylation in aromatase (7.5-fold), 20ß-HSD (3-fold), and FSHR (2-fold) genes. Hormone receptor investigation highlighted an escalating connection between transcriptional hyper-methylation and contamination levels. Additionally, our study revealed a positive correlation between oocyte growth and global DNA methylation, suggesting BPA's potential to modify DNA methylation in female paradise threadfins. This effect likely occurs through changes in hormone receptor expression, persisting throughout oocyte maturation. Notably, our research, the first of its kind in estuarine areas, confirmed BPA contamination in paradise threadfins, raising concerns about potential health risks for humans.

Quantum Communications Feasibility Tests over a UK-Ireland 224 km Undersea Link.

The future quantum internet will leverage existing communication infrastructures, including deployed optical fibre networks, to enable novel applications that outperform current information technology. In this scenario, we perform a feasibility study of quantum communications over an industrial 224 km submarine optical fibre link deployed between Southport in the United Kingdom (UK) and Portrane in the Republic of Ireland (IE). With a characterisation of phase drift, polarisation stability and the arrival time of entangled photons, we demonstrate the suitability of the link to enable international UK-IE quantum communications for the first time.

Tissue nanotransfection causes tumor regression by its effect on nanovesicle cargo that alters microenvironmental macrophage state.

Extracellular vesicles (EVs) are nanovesicles released by all eukaryotic cells. This work reports the first nanoscale fluorescent visualization of tumor-originating vesicles bearing an angiogenic microRNA (miR)-126 cargo. In a validated experimental model of lethal murine vascular neoplasm, tumor-originating EV delivered its miR-126 cargo to tumor-associated macrophages (TAMs). Such delivery resulted in an angiogenic (LYVE+) change of state in TAM that supported tumor formation. Study of the trafficking of tumor-originating fluorescently tagged EV revealed colocalization with TAM demonstrating uptake by these cells. Ex vivo treatment of macrophages with tumor-derived EVs led to gain of tumorigenicity in these isolated cells. Single-cell RNA sequencing of macrophages revealed that EV-borne miR-126 characterized the angiogenic change of state. Unique gene expression signatures of specific macrophage clusters responsive to miR-126-enriched tumor-derived EVs were revealed. Topical tissue nanotransfection (TNT) delivery of an oligonucleotide comprising an anti-miR against miR-126 resulted in significant knockdown of miR-126 in the tumor tissue. miR-126 knockdown resulted in complete involution of the tumor and improved survival rate of tumor-affected mice. This work identifies a novel tumorigenic mechanism that relies on tumorigenic state change of TAM caused by tumor-originating EV-borne angiomiR. This disease process can be effectively targeted by topical TNT of superficial tumors.

IDLat: An Importance-Driven Latent Generation Method for Scientific Data.

Deep learning based latent representations have been widely used for numerous scientific visualization applications such as isosurface similarity analysis, volume rendering, flow field synthesis, and data reduction, just to name a few. However, existing latent representations are mostly generated from raw data in an unsupervised manner, which makes it difficult to incorporate domain interest to control the size of the latent representations and the quality of the reconstructed data. In this paper, we present a novel importance-driven latent representation to facilitate domain-interest-guided scientific data visualization and analysis. We utilize spatial importance maps to represent various scientific interests and take them as the input to a feature transformation network to guide latent generation. We further reduced the latent size by a lossless entropy encoding algorithm trained together with the autoencoder, improving the storage and memory efficiency. We qualitatively and quantitatively evaluate the effectiveness and efficiency of latent representations generated by our method with data from multiple scientific visualization applications.

SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling.

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

Polyamine biosynthesis and eIF5A hypusination are modulated by the DNA tumor virus KSHV and promote KSHV viral infection.

Polyamines are critical metabolites involved in various cellular processes and often dysregulated in cancers. Kaposi's sarcoma-associated Herpesvirus (KSHV), a defined human oncogenic virus, leads to profound alterations of host metabolic landscape to favor development of KSHV-associated malignancies. In our studies, we identified that polyamine biosynthesis and eIF5A hypusination are dynamically regulated by KSHV infection through modulation of key enzymes (ODC1 and DHPS) of these pathways. During KSHV latency, ODC1 and DHPS are upregulated along with increase of hypusinated eIF5A (hyp-eIF5A), while hyp-eIF5A is further induced along with reduction of ODC1 and intracellular polyamines during KSHV lytic reactivation. In return these metabolic pathways are required for both KSHV lytic reactivation and de novo infection. Further analysis unraveled that synthesis of critical KSHV latent and lytic proteins (LANA, RTA) depends on hypusinated-eIF5A. We also demonstrated that KSHV infection can be efficiently and specifically suppressed by inhibitors targeting these pathways. Collectively, our results illustrated that the dynamic and profound interaction of a DNA tumor virus (KSHV) with host polyamine biosynthesis and eIF5A hypusination pathways promote viral propagation, thus defining new therapeutic targets to treat KSHV-associated malignancies.

Pathway-resolved decomposition demonstrates correlation and noise dependencies of redundant information processing in recurrent feed-forward topologies.

In a biochemical assay that converts fan-in networks into feed-forward loops (FFLs), we show that the inter-regulator redundant information about the output gene product can be decomposed into finer components, mediated by the constituent pathways. Variance-based information within the linear noise regime facilitates quantifying these submodular redundancies. Contrary to the conventional wisdom on information decomposition, we report that information redundancy depends nontrivially on inter-regulator correlation. For the type-1 coherent (C1) and incoherent (I1) FFLs, the direct regulatory path-mediated redundancy is certainly correlation independent. However, components induced by the indirect regulatory path and interpathway interference are correlation dependent in (non)linear fashion. The trade-off between information redundancy and similarly decomposable extrinsic noise from input to output node has been demonstrated for the pathways and full motifs. Our analyses suggest that the interpathway cross redundancy positively and negatively influences the superposition of elementary redundancies in the C1- and I1-FFLs, respectively. Their corresponding total extrinsic noise is produced by the weighted sum and difference of the pathway-specific components. We find that the I1-FFL is able to manufacture more varied redundancy and extrinsic noise responses compared to the C1-FFL. Underlying the differing characteristics of the composite metrics across FFL variants, there exist uniformly behaving pathway-dependent elements. The decomposition framework has been meticulously explored in biologically rational parametric realizations through analytical estimates and stochastic simulations.

Information transmission in a two-step cascade: interplay of activation and repression.

We present an information-theoretic formalism to study signal transduction in four architectural variants of a model two-step cascade with increasing input population. Our results categorize these four types into two classes depending upon the effect of activation and repression on mutual information, net synergy, and signal-to-noise ratio. Using the Gaussian framework and linear noise approximation, we derive the analytic expressions for these metrics to establish their underlying relationships in terms of the biochemical parameters. We also verify our approximations through stochastic simulations.

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination.

Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.

SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of antiviral interferon signaling.

FACT ( FA cilitates C hromatin T ranscription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

Probabilistic Data-Driven Sampling via Multi-Criteria Importance Analysis.

Although supercomputers are becoming increasingly powerful, their components have thus far not scaled proportionately. Compute power is growing enormously and is enabling finely resolved simulations that produce never-before-seen features. However, I/O capabilities lag by orders of magnitude, which means only a fraction of the simulation data can be stored for post hoc analysis. Prespecified plans for saving features and quantities of interest do not work for features that have not been seen before. Data-driven intelligent sampling schemes are needed to detect and save important parts of the simulation while it is running. Here, we propose a novel sampling scheme that reduces the size of the data by orders-of-magnitude while still preserving important regions. The approach we develop selects points with unusual data values and high gradients. We demonstrate that our approach outperforms traditional sampling schemes on a number of tasks.

Mitochondria as Target for Tumor Management of Hemangioendothelioma.

Aims: Hemangioendothelioma (HE) may be benign or malignant. Mouse hemangioendothelioma endothelial (EOMA) cells are validated to study mechanisms in HE. This work demonstrates that EOMA cells heavily rely on mitochondria to thrive. Thus, a combination therapy, including weak X-ray therapy (XRT, 0.5 Gy) and a standardized natural berry extract (NBE) was tested. This NBE is known to be effective in managing experimental HE and has been awarded with the Food and Drug Administration Investigational New Drug (FDA-IND) number 140318 for clinical studies on infantile hemangioma. Results: NBE treatment alone selectively attenuated basal oxygen consumption rate of EOMA cells. NBE specifically sensitized EOMA, but not murine aortic endothelial cells to XRT-dependent attenuation of mitochondrial respiration and adenosine triphosphate (ATP) production. Combination treatment, selectively and potently, influenced mitochondrial dynamics in EOMA cells such that fission was augmented. This was achieved by lowering of mitochondrial sirtuin 3 (SIRT3) causing increased phosphorylation of AMP-activated protein kinase (AMPK). A key role of SIRT3 in loss of EOMA cell viability caused by the combination therapy was evident when pyrroloquinoline quinone, an inducer of SIRT3, pretreatment rescued these cells. Innovation and Conclusion: Mitochondria-targeting NBE significantly extended survival of HE-affected mice. The beneficial effect of NBE in combination with weak X-ray therapy was, however, far more potent with threefold increase in murine survival. The observation that safe natural products may target tumor cell mitochondria and sharply lower radiation dosage required for tumor management warrants clinical testing.

Inhibition of Polo-like kinase 1 (PLK1) facilitates the elimination of HIV-1 viral reservoirs in CD4+ T cells ex vivo.

Although combination antiretroviral therapy is effective in controlling HIV-1 infection, latent HIV-1 proviruses cannot be eliminated. HIV-1 reactivation induced by the mere use of latency-reversing agents is insufficient to render death of reservoir cells, indicating that certain intrinsic survival mechanisms exist. We report that Polo-like kinase 1 (PLK1) plays a critical role in survival of CD4+ T cells that undergo HIV-1 reactivation from latency or de novo infection. PLK1 is elevated in both scenarios, which requires HIV-1 Nef. HIV-1 enhances PLK1 SUMOylation, causing its nuclear translocation and protein stabilization. Inhibition or knockdown of PLK1 markedly facilitates death of HIV-1-infected CD4+ T cells. Furthermore, PLK1 inhibitors strikingly reduce the size of HIV-1 latent reservoirs in primary CD4+ T cells. Our findings demonstrate that HIV-1 infection hijacks PLK1 to prevent cell death induced by viral cytopathic effects, and that PLK1 is a promising target for chemical "killing" of HIV-1 reservoir cells.

Profiling of immune related genes silenced in EBV-positive gastric carcinoma identified novel restriction factors of human gammaherpesviruses.

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses.

Extrinsic noise of the target gene governs abundance pattern of feed-forward loop motifs.

Feed-forward loop (FFL) is found to be a recurrent structure in bacterial and yeast gene transcription regulatory networks. In a generic FFL, transcription factor (TF) S regulates production of another TF X while both of these TFs regulate production of final gene-product Y. Depending upon the regulatory programs (activation or repression), FFLs are grouped into two broad classes: coherent (C) and incoherent (I), each class containing four distinct types (C1-C4 and I1-I4). These FFL types are experimentally observed to occur with varied frequencies, C1 and I1 being the abundant ones. Here we present a stochastic framework singling out the absolute value of the normalized covariance of X and Y to be the determining factor behind the abundance of FFLs while considering differential promoter activities of X and Y. Our theoretical construct employs two possible signal integration mechanisms (additive and multiplicative) to synthesize Y while steady-state population level of S remains fixed or becomes tunable reflecting two possible environmental signaling scenarios. Our model categorically points out that abundant FFLs exhibit higher amount of the designated metric which has a biophysical connotation of extrinsic noise for the target gene Y. Our predictions emanating from an overarching analytical expression utilizing biologically plausible parametric conditions are substantiated by stochastic simulation.

Isoniazid and Rifampicin Produce Hepatic Fibrosis through an Oxidative Stress-Dependent Mechanism.

METHODS: A combined dose of INH (50 mg) and RMP (100 mg) per kg body weight per day was administered to mice by oral gavage, 6 days a week, for 4 to 24 weeks for the assessment of liver injury, oxidative stress, and development of hepatic fibrosis, including demonstration of changes in key fibrogenesis linked pathways and mediators. RESULTS: Progressive increase in markers of hepatic stellate cell (HSC) activation associated with changes in matrix turnover was observed between 12 and 24 weeks of INH-RMP treatment along with the elevation of liver collagen content and significant periportal fibrosis. These were associated with concurrent apoptosis of the hepatocytes, increase in hepatic cytochrome P450 2E1 (CYP2E1), NADPH oxidase (NOX) activity, and development of hepatic oxidative stress. CONCLUSIONS: INH-RMP can activate HSC through generation of NOX-mediated oxidative stress, leading to the development of liver fibrosis.