Electrochemical DNA Biosensor That Detects Early Celiac Disease Autoantibodies.

Although it is estimated that more than one million Americans have celiac disease (CD), it remains challenging to diagnose. CD, an autoimmune and inflammatory response following the ingestion of gluten-containing foods, has symptoms overlapping with other diseases and requires invasive diagnostics. The gold standard for CD diagnosis involves serologic blood tests followed by invasive confirmatory biopsies. Here, we propose a less invasive method using an electrochemical DNA (E-DNA) biosensor for CD-specific autoantibodies (AABs) circulating in blood. In our approach, CD-specific AABs bind a synthetic neoepitope, causing a conformational change in the biosensor, as well as a change in the environment of an attached redox reporter, producing a measurable current reduction. We assessed the biosensor's ability to detect CD-specific patient-derived AABs in physiological buffer as well as buffer supplemented with bovine serum. Our biosensor was able to detect AABs in a dose-dependent manner; increased signal change correlated with increased AAB concentration with an apparent dissociation constant of 0.09 ± 0.03 units/mL of AABs. Furthermore, we found our biosensor to be target-specific, with minimal off-target binding of multiple unrelated biomarkers. Future efforts aimed at increasing sensitivity in complex media may build upon the biosensor design presented here to further improve CD AAB detection and CD diagnostic tools.

The challenges of closing an ileostomy in patients with total intestinal aganglionosis after small bowel transplant.

We present the case of a 14-year-old male with a history of small bowel transplantation for long segment Hirschsprung's disease who underwent Duhamel ileorectal pull-through procedure. In post-transplant, the patient had no restrictions and was not TPN-dependent. To improve his quality of life, he and his family were interested in closing the ileostomy and undergoing pull-through surgery. The complexity of the case includes the presence of an aganglionic rectal segment-a short root of the mesentery due to the small bowel transplant-and significant immunosuppression. At the moment, he is continent, doing well, and has not had any remarkable complications.

STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition.

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design.

Technical Variant Liver Transplant Utilization for Pediatric Recipients: Equal Graft Survival to Whole Liver Transplants and Promotion of Timely Transplantation Only When Performed at High-volume Centers.

BACKGROUND: Technical variant liver transplantation (TVLT) is a strategy to mitigate persistent pediatric waitlist mortality in the United States, although its implementation remains stagnant. This study investigated the relationship between TVLT utilization, transplant center volume, and graft survival. METHODS: Pediatric liver transplant recipients from 2010 to 2020 (n = 5208) were analyzed using the Scientific Registry of Transplant Recipients database. Transplant centers were categorized according to the average number of pediatric liver transplants performed per year (high-volume, ≥5; low-volume, <5). Graft survival rates were compared using Kaplan-Meier curves and log-rank tests. Cox proportional hazards models were used to identify predictors of graft failure. RESULTS: High-volume centers demonstrated equivalent whole liver transplant and TVLT graft survival ( P = 0.057) and significantly improved TVLT graft survival compared with low-volume centers ( P < 0.001). Transplantation at a low-volume center was significantly associated with graft failure (adjusted hazard ratio, 1.6; 95% confidence interval, 1.14-2.24; P = 0.007 in patients <12 y old and 1.8; 95% confidence interval, 1.13-2.87; P = 0.013 in patients ≥12 y old). A subset of high-volume centers with a significantly higher rate of TVLT use demonstrated a 23% reduction in waitlist mortality. CONCLUSIONS: Prompt transplantation with increased TVLT utilization at high-volume centers may reduce pediatric waitlist mortality without compromising graft survival.

Antiviral Therapy Utilization and 10-Year Outcomes in Resected Hepatitis B Virus- and Hepatitis C Virus-Related Hepatocellular Carcinoma.

PURPOSE: There are limited data on antiviral treatment utilization and its impact on long-term outcomes of hepatitis B virus (HBV)- and hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) after hepatic resection. We aimed to determine the utilization and impact of antivirals in HBV- and HCV-related HCC. METHODS: This cohort study included 1,906 participants (1,054 HBV-related HCC and 852 HCV-related HCC) from 12 international sites. All participants had HBV- or HCV-related HCC and underwent curative surgical resection. The primary outcome was the utilization of antiviral therapy, and the secondary outcome was long-term overall survival (OS). RESULTS: The mean (±standard deviation [SD]) age was 62.1 (±11.3) years, 74% were male, and 84% were Asian. A total of 47% of the total cohort received antiviral therapy during a mean (±SD) follow-up of 5.0 (±4.3) years. The overall antiviral utilization for participants with HBV-related HCC was 57% and declined over time, from 65% before 2010, to 60% from 2010 to 2015, to 47% beyond 2015, P < .0001. The overall utilization of antivirals for HCV-related HCC was 35% and increased over time, from 24% before 2015 to 74% from 2015 and beyond, P < .0001. The 10-year OS was lower in untreated participants for both HBV (58% v 61%) and HCV participants (38% v 82%; both P < .0001). On multivariable Cox regression analysis adjusted for relevant confounders, antiviral therapy initiated before or within 6 months of HCC diagnosis was independently associated with lower mortality in both HBV- (adjusted hazard ratio [aHR], 0.60 [95% CI, 0.43 to 0.83]; P = .002) and HCV-related HCC (aHR, 0.18 [95% CI, 0.11 to 0.31]; P < .0001). CONCLUSION: Antiviral therapy is associated with long-term survival in people with HBV- or HCV-related HCC who undergo curative resection but is severely underutilized.

Detection of IP-10 protein marker in undiluted blood serum via an electrochemical E-DNA scaffold sensor.

We describe an electrochemical analog of fluorescence polarization that supports the quantitative measurement of a specific protein, the chemokine IP-10, directly in undiluted blood serum. The sensor is label-free, wash-free, and electronic, suggesting it could support point-of-care detection of diagnostic proteins in largely unprocessed clinical samples.

Rapid and Quantitative Detection of Lung Cancer Biomarker ENOX2 Using a Novel Aptamer in an Electrochemical DNA-Based (E-DNA) Biosensor.

To overcome early cancer detection challenges, diagnostic tools enabling more sensitive, rapid, and noninvasive detection are necessary. An attractive cancer target for diagnostic blood tests is human Ecto-NOX disulfide-thiol exchanger 2 (ENOX2), expressed in most human cancer types and regularly shed into blood sera. Here, we developed an electrochemical DNA-based (E-DNA) biosensor that rapidly detects physiologically relevant levels of ENOX2. To identify ENOX2-binding aptamers that could potentially be used in a biosensor, recombinantly expressed ENOX2 was used as a binding target in an oligonucleotide library pull-down that generated a highly enriched ENOX2-binding aptamer. This candidate aptamer sensitively bound ENOX2 via gel mobility shift assays. To enable this aptamer to function in an ENOX2 E-DNA biosensor, the aptamer sequence was modified to adopt two conformations, one capable of ENOX2 binding, and one with disrupted ENOX2 binding. Upon ENOX2 introduction, a conformational shift to the ENOX2 binding state resulted in changed dynamics of a redox reporter molecule, which generated a rapid, significant, and target-specific electrical current readout change. ENOX2 biosensor sensitivity was at or below the diagnostic range. The ENOX2 E-DNA biosensor design presented here may enable the development of more sensitive, rapid, diagnostic tools for early cancer detection.

Quantification of transcription factor binding in cell extracts using an electrochemical, structure-switching biosensor.

Transcription factor expression levels, which sensitively reflect cellular development and disease state, are typically monitored via cumbersome, reagent-intensive assays that require relatively large quantities of cells. Here, we demonstrate a simple, quantitative approach to their detection based on a simple, electrochemical sensing platform. This sensor sensitively and quantitatively detects its target transcription factor in complex media (e.g., 250 µg/mL crude nuclear extracts) in a convenient, low-reagent process requiring only 10 µL of sample. Our approach thus appears a promising means of monitoring transcription factor levels.

The impact of hepatic portoenterostomy on liver transplantation for the treatment of biliary atresia: early failure adversely affects outcome.

The most common indication for pediatric LTx is biliary atresia with failed HPE, yet the effect of previous HPE on the outcome after LTx has not been well characterized. We retrospectively reviewed a single-center experience with 134 consecutive pediatric liver transplants for the treatment of biliary atresia from 1 May 1995 to 28 April 2008. Of 134 patients, 22 underwent LTx without prior HPE (NPE), while 112 patients underwent HPE first. HPE patients were grouped into EF, defined as need for LTx within the first year of life, and LF, defined as need for LTx beyond the first year of life. NPE and EF groups differed significantly from the LF group in age, weight, PELD, and ICU status (p < 0.05) with NPE having the highest PELD and ICU status. Patients who underwent salvage LTx after EF following HPE had a significantly higher incidence of post-operative bacteremia and septicemia (p < 0.05), and subsequently lower survival rates. One-year patient survival and graft survival were as follows: NPE 100%, EF 81%, and LF 96% (p < 0.05); and NPE 96%, EF 79%, and LF 96% (p < 0.05). Further investigation into the optimal treatment of biliary atresia should focus on identifying patients at high risk of EF who may benefit from proceeding directly to LTx given the increased risk of post-LTx bacteremia, sepsis, and death after failed HPE.

Differentiating Human Pluripotent Stem Cells to Cardiomyocytes Using Purified Extracellular Matrix Proteins.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

Transcription factor beacons for the quantitative detection of DNA binding activity.

The development of convenient, real-time probes for monitoring protein function in biological samples represents an important challenge of the postgenomic era. In response, we introduce here "transcription factor beacons," binding-activated fluorescent DNA probes that signal the presence of specific DNA-binding activities. As a proof of principle, we present beacons for the rapid, sensitive detection of three transcription factors (TATA Binding Protein, Myc-Max, and NF-κB), and measure binding activity directly in crude nuclear extracts.

Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays.

We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein complexes binding to DNA with varied specificity and affinity. Our approach is based on the novel coupling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double-stranded DNA microarrays and dye-labeled regulatory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experiment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF-PBM are similar to those determined by traditional methods, while simultaneous measurement of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF-PBM provides a novel and extendible platform for multi-protein transcription factor investigation.

Temperature and time-resolved total internal reflectance fluorescence analysis of reusable DNA hydrogel chips.

Total internal reflection fluorescence (TIRF) coupled with hydrogel-DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development.

Allele-specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene (TERT) associates with TERT expression in multiple cancers.

Telomerase reverse transcriptase (TERT) is pathologically expressed in the vast majority of human cancers, but the epigenetic regulation of its expression is only beginning to be understood. In particular, the active TERT gene in cancer cells has been characterized as having a hypermethylated CpG island, opposite to the general association of DNA methylation with gene repression. Here, we analyzed TERT promoter CpG methylation in 833 human cancer cell lines representing 23 different tissue types and found hypermethylation of the upstream portion of the CpG island and more conserved hypomethylation of a region including the proximal TERT promoter and exon 1. In cell lines with monoallelic expression of TERT, we found allelic methylation of the proximal TERT promoter. This included cell lines with the -124 or -146 activating promoter mutation as well as wild-type TERT cancer lines. In these cell line types, decreased proximal promoter methylation is associated with the active allele. Compared to cells with monoallelic expression of TERT, lines with biallelic expression of TERT had even lower methylation in the proximal TERT promoter. Thus, in cell lines from cancers of many different tissues, the TERT proximal promoter has canonical DNA methylation, with low methylation correlating with increased TERT expression.

Reagentless, electrochemical approach for the specific detection of double- and single-stranded DNA binding proteins.

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.

A paucity of knowledge regarding nontuberculous mycobacterial lipids compared to the tubercle bacillus.

All mycobacteria, including nontuberculous mycobacteria (NTM), synthesize an array of lipids including phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM). While absent from Mycobacterium tuberculosis (M. tb), glycopeptidolipids (GPL) are critical to the biology of NTM. M. tb and some NTM also synthesize trehalose-containing glycolipids and phenolic glycolipids (PGL), key membrane constituents with essential roles in metabolism. While lipids facilitate immune evasion, they also induce host immunity against tuberculosis. However, much less is known about the significance of NTM-derived PIM, LM, LAM, GPL, trehalose-containing glycolipids, and PGL as virulence factors, warranting further investigation. While culling the scientific literature on NTM lipids, it's evident that such studies were relatively few in number with the overwhelming majority of prior work dedicated to understanding lipids from the saprophyte Mycobacterium smegmatis. The identification and functional analysis of immune reactive NTM-derived lipids remain challenging, but such work is likely to yield a greater understanding of the pathogenesis of NTM lung disease. In this review, we juxtapose the vast literature of what is currently known regarding M. tb lipids to the lesser number of studies for comparable NTM lipids. But because GPL is the most widely recognized NTM lipid, we highlight its role in disease pathogenesis.

Single cell analysis of human foetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors.

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM+ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.

Gold Nanoparticle-Functionalized Reverse Thermal Gel for Tissue Engineering Applications.

Utilizing polymers in cardiac tissue engineering holds promise for restoring function to the heart following myocardial infarction, which is associated with grave morbidity and mortality. To properly mimic native cardiac tissue, materials must not only support cardiac cell growth but also have inherent conductive properties. Here, we present an injectable reverse thermal gel (RTG)-based cardiac cell scaffold system that is both biocompatible and conductive. Following the synthesis of a highly functionalizable, biomimetic RTG backbone, gold nanoparticles (AuNPs) were chemically conjugated to the backbone to enhance the system's conductivity. The resulting RTG-AuNP hydrogel supported targeted survival of neonatal rat ventricular myocytes (NRVMs) for up to 21 days when cocultured with cardiac fibroblasts, leading to an increase in connexin 43 (Cx43) relative to control cultures (NRVMs cultured on traditional gelatin-coated dishes and RTG hydrogel without AuNPs). This biomimetic and conductive RTG-AuNP hydrogel holds promise for future cardiac tissue engineering applications.

Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications.

Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.

Detection of sequence-specific protein-DNA interactions via surface enhanced resonance Raman scattering.

We developed a new strategy to detect protein-DNA binding through surface enhanced resonance Raman scattering (SERRS). Silver-plated DNA and gold nanoparticle assemblies were used to identify sequence-specific and concentration-dependent binding of a DNA cytosine-C5-methyltransferase and the eukaryotic transcriptional regulator, TATA binding protein. Proteins were identified through specific Raman-active labels, and affinities observed correlate well with those determined by other methods. Raman-active labeling and interchangeable DNA sequences create a platform for versatile investigation of multiprotein complexes.