Investigating Protein Binding with the Isothermal Ligand-induced Resolubilization Assay.

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.

Protein arginine N-methyltransferase 2 plays a noncatalytic role in the histone methylation activity of PRMT1.

Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.

Understanding the role of Pax5 in development of taxane-resistant neuroendocrine like prostate cancers.

Resistance to the current Androgen Receptor Signaling Inhibitor (ARSI) therapies has led to higher incidences of therapy-induced neuroendocrine-like prostate cancer (t-NEPC). This highly aggressive subtype with predominant small cell-like characteristics is resistant to taxane chemotherapies and has a dismal overall survival. t-NEPCs are mostly treated with platinum-based drugs with a combination of etoposide or taxane and have less selectivity and high systemic toxicity, which often limit their clinical potential. During t-NEPC transformation, adenocarcinomas lose their luminal features and adopt neuro-basal characteristics. Whether the adaptive neuronal characteristics of t-NEPC are responsible for such taxane resistance remains unknown. Pathway analysis from patient gene-expression databases indicates that t-NEPC upregulates various neuronal pathways associated with enhanced cellular networks. To identify transcription factor(s) (TF) that could be important for promoting the gene expression for neuronal characters in t-NEPC, we performed ATAC-Seq, acetylated-histone ChIP-seq, and RNA-seq in our NE-like cell line models and analyzed the promoters of transcriptionally active and significantly enriched neuroendocrine-like (NE-like) cancer-specific genes. Our results indicate that Pax5 could be an important transcription factor for neuronal gene expression and specific to t-NEPC. Pathway analysis revealed that Pax5 expression is involved in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are critical for strong cellular communications. Further results suggest that depletion of Pax5 disrupts cellular interaction in NE-like cells and reduces surface growth factor receptor activation, thereby, sensitizing them to taxane therapies. Moreover, t-NEPC specific hydroxymethylation of Pax5 promoter CpG islands favors Pbx1 binding to induce Pax5 expression. Based on our study, we concluded that continuous exposure to ARSI therapies leads to epigenetic modifications and Pax5 activation in t-NEPC, which promotes the expression of genes necessary to adopt taxane-resistant NE-like cancer. Thus, targeting the Pax5 axis can be beneficial for reverting their taxane sensitivity.

Contributions of flexor hallucis longus and brevis muscles to isometric toe flexor force production.

PURPOSE: Morphological differences between the two primary great toe flexors - flexor hallucis longus (FHL) and flexor hallucis brevis (FHB) - likely drive differences in how these muscles contribute to functional toe flexor torque production. The aim of the study was to investigate FHL and FHB activation in two isometric toe flexion tasks - one called a "toe-pushing" task with the metatarsophalangeal (MTP) joints dorsiflexed and the interphalangeal (IP) joints in neutral and another called a "toe-gripping" task with the MTP joints in neutral and flexed IP joints. METHODS: Twenty participants' FHL and FHB muscles were instrumented with intramuscular electromyography electrodes. Muscle activation was normalized to a maximum voluntary contraction and compared between the two isometric toe flexor force production tasks. RESULTS: Overall, participants utilized these two toe flexors completely differently in the two tasks. In the toe-gripping task, the FHL was activated to a much greater extent than the FHB. In fact, 18 our of 20 participants activated FHL at more than 70% maximum voluntary contraction and half of participants activated FHB at less than 10%. In contrast, muscle activation during the toe-pushing task appeared more reliant on the FHB for most participants. CONCLUSIONS: Different contributions from the FHL and FHB to toe flexor force production in these two tasks are potentially driven by differences in muscle functional length among other factors. These findings help to inform the selection of rehabilitation and training exercises meant to preferentially target intrinsic or extrinsic foot musculature.

Protein arginine N-methyltransferase activity determination with filter binding and phosphor screening (FBAPS) assay.

We developed a cost-effective assay to measure protein arginine N-methyltransferase (PRMT) activity in a medium-throughput manner by combining P81 filter binding and phosphor screening (FBAPS). Recombinantly-expressed PRMT1 and coactivator-associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of glycine- and arginine-rich (GAR) protein and polyA binding protein 1 (PABP1(437-488)) as substrates, respectively, and radiolabelled S-adenosyl-L-[methyl-14C]-methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signals were measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (KM, kcat) for enzymes and substrates were determined, and IC50 values were obtained for well-characterized inhibitors. FBAPS yielded kinetic parameters with no statistically significant difference to what was obtained using liquid scintillation counting. The IC50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature. The FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for processing of samples in a multi-well format, moderately increasing throughput. Signal detection by phosphor imaging offers an affordable and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy.

Comparing global and local maps of the Caribbean pine forests of Andros, home of the critically endangered Bahama Oriole.

Forest loss is occurring at alarming rates across the globe. The pine rockland forests of Andros, The Bahamas, likely represent some of the largest stands of Bahamian subspecies of Caribbean pine in the world. Given the unique species that inhabit these pine forests, such as the endemic and critically endangered Bahama Oriole, monitoring habitats on Andros is crucial to inform conservation planning. We developed a 2019 land classification map to assess the status of nine terrestrial habitats on Andros. Our Random Forest classification model predicted habitat classes with high overall accuracy. Caribbean pine was the dominant land class making up roughly one-third of the total terrestrial area. Whereas much of the pine forest area was found as small patches, most were close to other patches of pine suggesting isolation of forest patches is low. We compared our known intact forest areas to recent forest loss identified by the Hansen et al. Global Forest Change product and assessed areas of habitat disturbance in high-resolution imagery. Our results suggest that this global map overpredicted forest loss on Andros. The small degree of true forest loss on Andros was driven mostly by anthropogenic activity. A cross-tabulation of the Hansen forest loss with fire data showed that understory fires were frequently associated with falsely classified deforestation. Given the threats of climate change to this open forest type-intensifying fire regimes, strengthening hurricanes, and sea level rise-monitoring changes in open forest extent is a critical task across the Caribbean region and the world.

Rapid Development of De Novo Thoracic Aneurysm After Liver Transplant.

The development of aortic aneurysms in post-transplant patients is a rare but potentially lethal problem. De novo aortic aneurysm formation and rapid growth are postulated to result from an imbalance between pro- and anti-inflammatory vascular endothelial factors after transplant. Here, we present a case of de novo thoracic aneurysm formation within 2 months of orthotopic liver transplant. Prompt clinical recognition allowed for successful endovascular repair. Transplant clinicians should be aware of this potentially life-threatening complication and monitor at-risk recipients accordingly.

Effect of a task's postural demands on medial longitudinal arch deformation and activation of foot intrinsic and extrinsic musculatur.

PURPOSE: It is not well established how motion and muscle activation of the medial longitudinal arch (MLA) of the foot vary under different loading conditions. Intrinsic and extrinsic foot muscles may play a role in postural control, which may be investigated by comparing loading tasks with differing postural demands. The objective of this study was to investigate the interaction of MLA flexibility and loading task on muscle activation. METHODS: Twenty healthy adults completed two instrumented single-foot loading tasks: controlled external load of 50% body weight while sitting and bilateral standing. Fine-wire intramuscular and surface electromyography collected flexor hallucis brevis, abductor hallucis, tibialis posterior, flexor hallucis longus, tibialis anterior, and peroneus longus activation. MLA deformation was measured as a percent change in navicular height with loading. RESULTS: During seated external loading, greater MLA deformation was associated with greater muscle activation for all instrumented muscles (R² = 0.224-0.303, p < 0.05) except for tibialis anterior. During bilateral stance, there were no correlations between MLA deformation and muscle activation. Activation of all extrinsic muscles except for tibialis anterior were greater during bilateral standing than during external loading ( p = 0.002-0.013), indicating activation of these muscles was caused by postural demands of the standing task, not simply load. CONCLUSIONS: MLA deformation and muscle activation are strongly task-dependent.

Utility of Transjugular Intrahepatic Portosystemic Shunt Placement for Maintaining Portal Vein Patency in Candidates on Wait Lists Who Develop Thrombus.

OBJECTIVES: Although no longer a contraindication to liver transplant, portal vein thrombosis may lead to longer operative time and complexities in venous reconstruction. Strategies to maintain preoperative patency include systemic anticoagulation and/or transjugular intrahepatic portosystemic shunt placement. The former may not be ideal in cirrhotic patients prone to luminal gastrointestinal tract bleeding, and factors that predict improvements in portal vein thrombosis with the latter have not been well defined. Our goal was to evaluate the effectiveness of transjugular intrahepatic portosystemic shunt placement as monotherapy to improve and/or resolve portal vein thrombosis in otherwise eligible liver transplant candidates with partial or complete portal vein thrombosis and to identify factors predicting success. MATERIALS AND METHODS: We identified 30 patients from 2010 to 2015 who had transjugular intrahepatic portosystemic shunt placementfor primary indication to maintain portal vein patency. RESULTS: The main portal vein was completely thrombosed in 5 of 30 (16.6%), nearly completely thrombosed in 9 of 30 (30%), and partially thrombosed in 16 patients (53.3%). Twenty-four patients (80%) had improvement and/or resolution of portal vein thrombosis after transjugular intrahepatic portosystemic shunt placement, with 18 of these (75%) having complete resolution. All 5 patients (20.8%) with complete thrombosis had improvement/resolution of portal vein thrombosis. Nine patients (30%) required hospitalization within 3 months for hepatic encephalopathy. There were 3 deaths (10%) not related to transjugular intrahepatic portosystemic shunt placement (10%). Nine patients underwent liver transplant after shunt placement (median 2.9 mo; range, 0.3-32 mo); all 9 received endto-end anastomosis without need for intraoperative thrombectomy. CONCLUSIONS: Transjugular intrahepatic portosystemic shunt placement may be effective as monotherapy for maintaining or restoring portal vein patency in selected livertransplant candidates, even in those with complete portal vein thrombosis. Further studies are needed to define potential responders to this approach.

Structure-function studies of tetrahydroprotoberberine N-methyltransferase reveal the molecular basis of stereoselective substrate recognition.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse class of plant-specialized metabolites that have been particularly well-studied in the order Ranunculales. The N-methyltransferases (NMTs) in BIA biosynthesis can be divided into three groups according to substrate specificity and amino acid sequence. Here, we report the first crystal structures of enzyme complexes from the tetrahydroprotoberberine NMT (TNMT) subclass, specifically for GfTNMT from the yellow horned poppy (Glaucium flavum). GfTNMT was co-crystallized with the cofactor S-adenosyl-l-methionine (dmin = 1.6 Å), the product S-adenosyl-l-homocysteine (dmin = 1.8 Å), or in complex with S-adenosyl-l-homocysteine and (S)-cis-N-methylstylopine (dmin = 1.8 Å). These structures reveal for the first time how a mostly hydrophobic L-shaped substrate recognition pocket selects for the (S)-cis configuration of the two central six-membered rings in protoberberine BIA compounds. Mutagenesis studies confirm and functionally define the roles of several highly-conserved residues within and near the GfTNMT-active site. The substrate specificity of TNMT enzymes appears to arise from the arrangement of subgroup-specific stereospecific recognition elements relative to catalytic elements that are more widely-conserved among all BIA NMTs. The binding mode of protoberberine compounds to GfTNMT appears to be similar to coclaurine NMT, with the isoquinoline rings buried deepest in the binding pocket. This binding mode differs from that of pavine NMT, in which the benzyl ring is bound more deeply than the isoquinoline rings. The insights into substrate recognition and catalysis provided here form a sound basis for the rational engineering of NMT enzymes for chemoenzymatic synthesis and metabolic engineering.

Immune Reconstitution Syndrome with Initiation of Treatment of HBV/HIV Co-infection: Activity Flare associated with E antigen Seroconversion.

Immune reconstitution syndrome is a recognized complication with initiation of highly active antiretroviral therapy for acquired immune deficiency syndrome patients co-infected with hepatitis B. Hepatitis B flares are seen in 20%-25% of patients after initiation of highly active antiretroviral therapy, an estimated 1%-5% of whom develop clinical hepatitis. We present a case of highly active antiretroviral therapy initiation for HIV that led to a flare of HBV activity despite antiviral therapy directed towards both. Liver biopsy and longitudinal serologic evaluation lend support to the hypothesis that the flare in activity was representative of IRIS. Importantly, we document eAg/eAb seroconversion with the IRIS phenomenon.

Real-Time Ultrasound-Guided Paracentesis by Radiologists: Near Zero Risk of Hemorrhage without Correction of Coagulopathy.

PURPOSE: To evaluate the rate and risk factors for hemorrhage in patients undergoing real-time, ultrasound-guided paracentesis by radiologists without correction of coagulopathy. MATERIALS AND METHODS: This was a retrospective study of all patients who underwent real-time, ultrasound-guided paracentesis at a single institution over a 2-year period. In total, 3116 paracentesis procedures were performed: 757 (24%) inpatients and 2,359 (76%) outpatients. Ninety-five percent of patients had a diagnosis of cirrhosis. Mean patient age was 56.6 years. Mean international normalized ratio (INR) was 1.6; INR was > 2 in 437 (14%) of cases. Mean platelet count was 122 x 103/µL; platelet count was < 50 x 103/µL in 368 (12%) of patients. Seven hundred seven (23%) patients were dialysis dependent. Patients were followed for 2 weeks after paracentesis to assess for hemorrhage requiring transfusion or rescue angiogram/embolization. Univariate analysis was performed to determine risk factors for hemorrhage. Blood product and cost saving analysis were performed. RESULTS: Significant post-paracentesis hemorrhage occurred in 6 (0.19%) patients, and only 1 patient required an angiogram with embolization. No predictors of post-procedure bleeding were found, including INR and platelet count. Transfusion of 1125 units of fresh frozen plasma and 366 units of platelets were avoided, for a transfusion-associated cost savings of $816,000. CONCLUSIONS: Without correction of coagulation abnormalities with prophylactic blood product transfusion, post-procedural hemorrhage is very rare when paracentesis is performed with real-time ultrasound guidance by radiologists.

Race and Gradient Difference Are Associated with Increased Risk of Hepatic Encephalopathy Hospital Admission After Transjugular Intrahepatic Portosystemic Shunt Placement.

BACKGROUND/AIMS: Hepatic encephalopathy (HE) is a well-recognized complication of transjugular intrahepatic portosystemic shunt (TIPS) placement. The aim of this investigation was to evaluate incidence and predictors of post-TIPS HE necessitating hospital admission in a non-clinical trial setting. METHODS: We performed a retrospective cohort study identifying 273 consecutive patients undergoing TIPS from 2010 to 2015 for any indication; 210 met inclusion/exclusion criteria. The primary endpoint was incidence of post-TIPS HE defined as encephalopathy with no other identifiable cause requiring hospitalization within 90 days of TIPS. Clinical demographics and procedural variables were collected and analyzed to determine predictors of readmission for post-TIPS HE. Categorical variables were analyzed using Fisher's exact test; continuous variables were compared using Levene's t-test and student's t-test; P < 0.05, significant. RESULTS: Forty-two of 210 patients (20%) developed post-TIPS HE requiring hospitalization within 90 days. On analysis of cohorts (post-TIPS HE vs. no post-TIPS HE): non-white race (31.0% vs. 17.5%, P = 0.022) and increased hepatic venous pressure gradient (HVPG) difference during TIPS (10.5 vs. 8.9 mmHg, P = 0.030) were associated with an increased incidence of HE requiring readmission within 90 days. CONCLUSIONS: HE remains a common complication of TIPS. Non-Caucasian race is a significant clinical demographic associated with increased risk for readmission. Independent of initial or final HVPG, HVPG difference appears to be a significant modifiable technical risk factor. In the absence of clear preventative strategies for post-TIPS encephalopathy, non-Caucasians with HVPG reductions >9 mmHg may require targeted follow up evaluation to prevent hospital readmission.

Refractory Hepatic Encephalopathy After Elective Transjugular Intrahepatic Portosystemic Shunt: Risk Factors and Outcomes with Revision.

BACKGROUND AND AIMS: Hepatic encephalopathy (HE) is a common complication of elective transjugular intrahepatic portosystemic shunt (TIPS) placement and is often successfully medically managed. Risk factors for refractory hepatic encephalopathy (RHE) necessitating revision of TIPS are not well defined. We evaluated the incidence, predictors, and outcomes of post-TIPS RHE necessitating TIPS revision. METHODS: In a retrospective cohort study of 174 consecutive patients undergoing elective TIPS placement (2010-2015), we evaluated the incidence of post-TIPS RHE. Clinical demographics and procedural variables were collected. 1-year outcomes after revision were collected. RESULTS: Ten of 174 patients (5.7%) developed post-TIPS RHE requiring revision. Significant differences between RHE and non-refractory groups were shunt size > 8 versus ≤ 8 mm (18.5 vs. 3.4%, p = 0.001), history of HE (14 vs. 2%, p = 0.007), and serum albumin levels ≤ 2.5 versus > 2.5 g/dL (13.1 vs. 3.1%, p = 0.020). On multivariate analysis, shunt size > 8 mm (p = 0.001), history of HE prior to TIPS (p = 0.006), and low serum albumin (≤ 2.5 g/dL) (p = 0.022) remained independent predictors of RHE, controlling for age and Model for End-Stage Liver Disease score. RHE improved in 8 of 10 patients but survival at 1 year without liver transplantation (LT) was only 10%. CONCLUSION: While TIPS revision successfully improves RHE in most cases, 1-year mortality rates are high, limiting the value of revision in non-LT candidates. Patients with previous history of HE and low serum albumin levels prior to TIPS may benefit most from the use of shunt sizes < 8 mm to mitigate the risk of RHE. LEVEL OF EVIDENCE: Level 4, case series.

Niraparib: A Poly(ADP-ribose) Polymerase (PARP) Inhibitor for the Treatment of Tumors with Defective Homologous Recombination.

Poly(ADP-ribose) polymerases (PARPs) are involved in DNA repair following damage by endogenous or exogenous processes. It has become clear over the past decade that inhibition of PARP in the context of defects in other DNA repair mechanisms provide a tumor specific way to kill cancer cells. We describe the rationale for this approach and the design and discovery of niraparib, a potent PARP-1/2 inhibitor with good cell based activity, selectivity for cancer over normal cells, and oral bioavailability. Niraparib was characterized in a number of preclinical models before moving to phase I clinical trials, where it showed excellent human pharmacokinetics suitable for once a day oral dosing, achieved its pharmacodynamic target for PARP inhibition, and had promising activity in cancer patients. It is currently being tested in phase 3 clinical trials as maintenance therapy in ovarian cancer and as a treatment for breast cancer.

Development of macrocyclic inhibitors of HCV NS3/4A protease with cyclic constrained P2-P4 linkers.

A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.

Development of potent macrocyclic inhibitors of genotype 3a HCV NS3/4A protease.

A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.

Synthesis and antiviral properties of novel tetracyclic nucleoside inhibitors of hepatitis C NS5B polymerase.

As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV NS5B polymerase, we report the discovery of a novel series of 2'-C-Methyl-ribose nucleoside derivatives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs) was established, enabling a solid understanding of the SAR relationship within the series. By this approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cellular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC(50) = 0.1 µM, a level of intrinsic potency similar to that of previous and current clinical candidates.