Selective internal radiation therapy with yttrium-90 resin microspheres followed by anatomical hepatectomy: A potential curative strategy in advanced hepatocellular carcinoma.

About 80% of hepatocellular carcinoma (HCC) patients are in advanced stages and ineligible for curative surgery. Palliative treatments just maintained limited survival, thus an effective downstaging therapy is badly needed. Here we report an initially unresectable patient who underwent radical hepatectomy after successful downstaging with selective internal radiation therapy (SIRT). A 34-year-old man was diagnosed with China Liver Cancer Staging (CNLC) IIIa HCC. Due to insufficient future liver remnant and vascular involvement, the patient was suggested to be unresectable. SIRT with yttrium-90 resin microspheres was given. At three months post-SIRT, a complete response was achieved. The tumor was downstaged to CNLC Ia stage. The patient underwent anatomical hepatectomy 5 months after SIRT. Histopathological examination of the resected specimen showed 4% viable tumor cells inside a necrotic mass. To our knowledge, this is the first case who underwent SIRT with yttrium-90 resin microspheres in China mainland. The success of the downstaging in this case renders a possible cure to be achieved in an initially unresectable patient. In addition, the nearly complete tumor necrosis in the resected specimen indicates a good prognosis post-surgery. This is the first case who underwent SIRT with yttrium-90 resin microspheres in China mainland. SIRT followed by anatomical hepatectomy is a potentially curative strategy for unresectable HCC, which deserves a confirmative trial in the future.

Implantation of a toric intraocular lens after repeated radial keratotomy procedures: A case report.

Corneal alterations due to radial keratotomy (RK) complicate intraocular lens calculations, which may explain why there have been few reports of toric intraocular lens (TIOL) implantation after excessive or multiple operations. A 71-year-old male with a history of repeated RKs and at least 30 corneal incisions in each eye was referred for cataract surgery. Preoperatively, the best-corrected distance visual acuity was 0.7 decimal (0.15 logMAR) in the right eye and 0.9 decimal (0.05 logMAR) in the left eye. The refractive errors were -8.00 -3.00 × 80 and -6.00 -3.50 × 80, respectively. The total corneal cylindrical powers (real power; anterior and posterior) were, respectively, -0.90 D and -3.60 D at 9 a.m., compared to -1.60 D and -3.80 D at 1 p.m. Corneal astigmatism in the left eye was considered symmetric and diurnally stable; therefore, an XY1AT6 TIOL (Hoya, Tokyo, Japan; cylindrical power at the plane, +3.75 D) was implanted. A non-toric intraocular lens, the XY1 (Hoya), was implanted in the right eye. Six-month postoperative best-corrected distance visual acuities were 1.2 decimal (-0.08 logMAR) and 1.0 decimal (0.00 logMAR) in the right and left eyes, respectively. Post-operative manifest refractions were +0.00 -3.00 × 70 and -1.00 -2.00 × 85, respectively. The TIOL reduced refractive astigmatism in the left eye; therefore, we believe that even after multiple RKs, the TIOL can be a suitable candidate to correct astigmatism if the corneal astigmatism is diurnally stable and symmetric.

NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications.

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

Development of an Electronic Medical Tool to Facilitate Allocation of Limited Resources in Times of Crisis.

The COVID-19 pandemic has made decisions about resource allocation and reallocation real possibilities even in high-resource settings. In April 2020, in preparation for such an eventuality, Atlantic Health System began to develop a real-time instrument built into the EMR to assist with such decisions. The instrument calculated the modified Sequential Organ Failure Assessment for all patients admitted, in real time, to assist triage teams make decisions if crisis standards of care were declared. A pilot assessment of the instrument was performed using retrospective data by nine members of the triage teams, who were asked to identify the six patients at highest risk of reallocation. Agreement about which patients were at highest risk of resource reallocation was good, but not perfect. All raters agreed on five of the six patients, but only seven of nine agreed on the final patient. Among the six consensus selections for reallocation, five died prior to hospital discharge. All patients at highest risk of reallocation had a predicted life expectancy of less than 1 year. In conclusion, the instrument was easy to use, and the concordance among raters was good but not perfect. Predicted life expectance was a major determinant of the triage score.

Emerging roles of interferon-stimulated genes in the innate immune response to hepatitis C virus infection.

Infection with hepatitis C virus (HCV), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. HCV infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. Upon HCV infection, the host induces the interferon (IFN)-mediated frontline defense to limit virus replication. Conversely, HCV employs diverse strategies to escape host innate immune surveillance. Type I IFN elicits its antiviral actions by inducing a wide array of IFN-stimulated genes (ISGs). Nevertheless, the mechanisms by which these ISGs participate in IFN-mediated anti-HCV actions remain largely unknown. In this review, we first outline the signaling pathways known to be involved in the production of type I IFN and ISGs and the tactics that HCV uses to subvert innate immunity. Then, we summarize the effector mechanisms of scaffold ISGs known to modulate IFN function in HCV replication. We also highlight the potential functions of emerging ISGs, which were identified from genome-wide siRNA screens, in HCV replication. Finally, we discuss the functions of several cellular determinants critical for regulating host immunity in HCV replication. This review will provide a basis for understanding the complexity and functionality of the pleiotropic IFN system in HCV infection. Elucidation of the specificity and the mode of action of these emerging ISGs will also help to identify novel cellular targets against which effective HCV therapeutics can be developed.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.

Amylin: Pharmacology, Physiology, and Clinical Potential.

Amylin is a pancreatic ß-cell hormone that produces effects in several different organ systems. Here, we review the literature in rodents and in humans on amylin research since its discovery as a hormone about 25 years ago. Amylin is a 37-amino-acid peptide that activates its specific receptors, which are multisubunit G protein-coupled receptors resulting from the coexpression of a core receptor protein with receptor activity-modifying proteins, resulting in multiple receptor subtypes. Amylin's major role is as a glucoregulatory hormone, and it is an important regulator of energy metabolism in health and disease. Other amylin actions have also been reported, such as on the cardiovascular system or on bone. Amylin acts principally in the circumventricular organs of the central nervous system and functionally interacts with other metabolically active hormones such as cholecystokinin, leptin, and estradiol. The amylin-based peptide, pramlintide, is used clinically to treat type 1 and type 2 diabetes. Clinical studies in obesity have shown that amylin agonists could also be useful for weight loss, especially in combination with other agents.

Effects of pramlintide in patients with type 2 diabetes mellitus: an analysis using daily insulin dose tertiles.

OBJECTIVE: The purpose of the analysis was to investigate if the efficacy and tolerability of 6 months of pramlintide therapy in patients with type 2 diabetes mellitus (T2DM) differed with increasing levels of concomitant insulin doses, using data from 3 previously described clinical trials. METHODS: In this post hoc analysis, data from 2 pooled, placebo-controlled pivotal trials and 1 clinical practice trial were evaluated by baseline insulin use tertile in patients with T2DM. RESULTS: In the pivotal trials, both glycated hemoglobin (A1C) and body weight decreased similarly across tertiles with pramlintide. A1C decreased slightly and body weight remained relatively unchanged across tertiles with placebo. Similarly, in the clinical practice trial, pramlintide was associated with decreases in A1C, body weight, and total daily insulin use across the tertiles. Overall, the most common adverse events were gastrointestinal in nature, and the rate of severe hypoglycemia was low. CONCLUSION: These results suggest that pramlintide therapy was associated with improved A1C and decreased body weight, with a low rate of severe hypoglycemia, among patients with T2DM, regardless of baseline insulin use.

Autophagy in hepatitis C virus-host interactions: potential roles and therapeutic targets for liver-associated diseases.

Autophagy is a lysosome-associated, degradative process that catabolizes cytosolic components to recycle nutrients for further use and maintain cell homeostasis. Hepatitis C virus (HCV) is a major cause of chronic hepatitis, which often leads to end-stage liver-associated diseases and is a significant burden on worldwide public health. Emerging lines of evidence indicate that autophagy plays an important role in promoting the HCV life cycle in host cells. Moreover, the diverse impacts of autophagy on a variety of signaling pathways in HCV-infected cells suggest that the autophagic process is required for balancing HCV-host cell interactions and involved in the pathogenesis of HCV-related liver diseases. However, the detailed molecular mechanism underlying how HCV activates autophagy to benefit viral growth is still enigmatic. Additionally, how the autophagic response contributes to disease progression in HCV-infected cells remains largely unknown. Hence, in this review, we overview the interplay between autophagy and the HCV life cycle and propose possible mechanisms by which autophagy may promote the pathogenesis of HCV-associated chronic liver diseases. Moreover, we outline the related studies on how autophagy interplays with HCV replication and discuss the possible implications of autophagy and viral replication in the progression of HCV-induced liver diseases, e.g., steatosis and hepatocellular carcinoma. Finally, we explore the potential therapeutics that target autophagy to cure HCV infection and its related liver diseases.

Hepatic arterioportal fistula presenting as gastric variceal hemorrhage.

Hepatic arterioportal fistulae (APF) are abnormal communications between the hepatic artery and the portal vein. In this report, we present the second case in the literature of a symptomatic APF presenting as a gastric variceal bleeding. A 55-year-old female presented to our facility with hematemesis. Upper endoscopy revealed a bleeding gastric varix. A computed tomography scan identified a large left hepatic lobe APF between the left hepatic artery and the left portal vein. Through angiography coil embolization was performed and with resultant loss of arterial flow, the APF was decompressed. On hospital day 3, the patient developed new melena. Portovenogram was performed and a TIPS stent was deployed. The patient subsequently did well. Hepatic arterioportal fistulae can result in portal hypertension secondary to arterial blood flowing directly into the portal vein bypassing the hepatic sinusoids. Iatrogenic causes (e.g. percutaneous liver biopsy) represent more than 50% of published cases of APFs. Most APFs resolve spontaneously as they are small and peripherally located. In rare instances, when APFs are centrally located, clinical symptoms develop. There have been 30 reported cases of symptomatic intrahepatic APFs following percutaneous liver biopsy. Of those, only one case presented as a gastric variceal bleed. Digital subtraction angiography is the gold standard in the diagnosis and treatment of APFs. In addition to initial embolization, we elected to treat the patient with TIPS due to the magnitude of her bleed. Although rare, intrahepatic APF should be kept on the differential of a patient presenting with isolated gastric varices.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Hepatitis C virus and cellular stress response: implications to molecular pathogenesis of liver diseases.

Infection with hepatitis C virus (HCV) is a leading risk factor for chronic liver disease progression, including steatosis, cirrhosis, and hepatocellular carcinoma. With approximately 3% of the human population infected worldwide, HCV infection remains a global public health challenge. The efficacy of current therapy is still limited in many patients infected with HCV, thus a greater understanding of pathogenesis in HCV infection is desperately needed. Emerging lines of evidence indicate that HCV triggers a wide range of cellular stress responses, including cell cycle arrest, apoptosis, endoplasmic reticulum (ER) stress/unfolded protein response (UPR), and autophagy. Also, recent studies suggest that these HCV-induced cellular responses may contribute to chronic liver diseases by modulating cell proliferation, altering lipid metabolism, and potentiating oncogenic pathways. However, the molecular mechanism underlying HCV infection in the pathogenesis of chronic liver diseases still remains to be determined. Here, we review the known stress response activation in HCV infection in vitro and in vivo, and also explore the possible relationship of a variety of cellular responses with the pathogenicity of HCV-associated diseases. Comprehensive knowledge of HCV-mediated disease progression shall shed new insights into the discovery of novel therapeutic targets and the development of new intervention strategy.

High-content screening data management for drug discovery in a small- to medium-size laboratory: results of a collaborative pilot study focused on user expectations as indicators of effectiveness.

High-content screening (HCS) technology provides a powerful vantage point to approach biological problems; it allows analysis of cell parameters, including changes in cell or protein movement, shape, or texture. As part of a collaborative pilot research project to improve bioscience research data integration, we identified HCS data management as an area ripe for advancement. A primary goal was to develop an integrated data management and analysis system suitable for small- to medium-size HCS programs that would improve research productivity and increase work satisfaction. A system was developed that uses Labmatrix, a Web-based research data management platform, to integrate and query data derived from a Cellomics STORE database. Focusing on user expectations, several barriers to HCS productivity were identified and reduced or eliminated. The impact of the project on HCS research productivity was tested through a series of 18 lab-requested integrated data queries, 7 of which were fully enabled, 7 partially enabled, and 4 enabled through data export to standalone data analysis tools. The results are limited to one laboratory, but this pilot suggests that through an "implementation research" approach, a network of small- to medium-size laboratories involved in HCS projects could achieve greater productivity and satisfaction in drug discovery research.

GLP-1R and amylin agonism in metabolic disease: complementary mechanisms and future opportunities.

The discoveries of the incretin hormone glucagon-like peptide-1 (GLP-1) and the ß-cell hormone amylin have translated into hormone-based therapies for diabetes. Both classes of molecules also exhibit weight-lowering effects and have been investigated for their anti-obesity potential. In the present review, we explore the mechanisms underlying the physiological and pharmacological actions of GLP-1 and amylin agonism. Despite their similarities (e.g. both molecular classes slow gastric emptying, decrease glucagon and inhibit food intake), there are important distinctions between the central and/or peripheral pathways that mediate their effects on glycaemia and energy balance. We suggest that understanding the similarities and differences between these molecules holds important implications for the development of novel, combination-based therapies, which are increasingly the norm for diabetes/metabolic disease. Finally, the future of GLP-1- and amylin agonist-based therapeutics is discussed.

Outreach in surgery at the undergraduate level: an opportunity to improve surgical interest among women?

Medical career choice is often formed at the premedical level, thus surgeons must reach out to undergraduates to enhance interest in surgery. Because there is a predominance of women among undergraduates (57%), this outreach also serves as an opportunity to introduce women to a surgical career. We developed an undergraduate course ("Surgery 99") offering course credit for participation in clinical research projects in surgery, shadowing surgeons in the operating room, and receiving mentorship for a surgical career. Six surgeons (50% women) served as course instructors. The final exam was a thesis with oral presentation. For enrollment, 132 students applied and 13 were accepted each quarter. Eleven students (85%) were women. None of the students had prior exposure to surgery. All but one student (93%) found the experience met or exceeded their expectations. Upon exit, knowledge attained was ranked highest, followed by observation in the operating room, and clinical research experience. All found that the course affirmed their decision to attend medical school and promoted their interest in surgery residency. We demonstrate a successful model for outreach in surgery at the undergraduate level that can positively influence interest in a surgical career, especially among women.

Identification of hnRNPH1, NF45, and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein.

The hepatitis C virus core protein (HCVc) forms the viral nucleocapsid and is involved in viral persistence and pathogenesis, possibly by interacting with host factors to modulate viral replication and cellular functions. Here, we identified 36 cellular protein candidates by one-dimensional SDS-PAGE and LC-MS/MS-based proteomics after affinity purification with HCVc174, a matured form of HCVc from HCV-1b genotype, tagged with biotin and calmodulin-binding peptide/protein A at N- and C-termini, respectively. By pull-down and confocal imaging techniques, we confirmed that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1), nuclear factor 45 (NF45), and C14orf166 are novel HCVc174-interacting host proteins, known to participate in mRNA metabolism, gene regulation, and microtubule organization, respectively. Unlike the other 2 proteins, NF45 interacted with HCVc174 in an RNA-dependent manner. These 3 proteins colocalized with ectopic HCVc-1b in both the cytoplasm and nucleus, which demonstrated their spatial interaction with naturally translocated HCVc174 after HCVc biogenesis. Such colocalization, however, shifted to the cytoplasm in cells with replicating virus of 1b or 2a genotype, indicating that active viral replication confined these interacting proteins in the cytoplasm. Collectively, our findings suggest that spatial interactions of hnRNPH1, NF45, and C14orf166 with HCVc174 likely modulate HCV or cellular functions during acute and chronic HCV infection.

An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system.

We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins.

Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro.

Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-ß activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.