Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri.

The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein-centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein-coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., "self-activating" behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.

A League of Its Own? Established and Emerging Therapies in Undifferentiated Pleomorphic Sarcoma.

OPINION STATEMENT: Over the last decade in soft tissue sarcoma (STS) research, the shifting landscape towards more precise subtype classification and the increasing study of novel therapeutic strategies has prompted a need to highlight current knowledge of effective subtype specific therapies. Undifferentiated pleomorphic sarcoma (UPS), formerly known as malignant fibrous histiocytoma (MFH), is among the most common subtypes of STS arising in the trunk or extremities of adults. Administration of systemic chemotherapy is the primary management in locally advanced and metastatic UPS. While anthracycline-based chemotherapy continues to be standard of care in this setting, outcomes in locally advanced or metastatic UPS remain poor. Recent studies highlight the unique characteristics of UPS that may contribute to its greater sensitivity to immune checkpoint inhibition (ICI) compared to other STS subtypes. With the promise of benefit from novel therapies, including ICI or ICI plus chemotherapy, for a subset of patients with UPS comes the need to identify biomarkers predictive of response to therapy. Ongoing and future clinical trials should place strong emphasis on correlative biomarker studies to learn more about the unique biology of UPS and to identify patients for whom ICI-based therapy will be effective.

A Case of Microsatellite Instability-High Colon Cancer in a Young Woman With Familial Adenomatous Polyposis.

Two major molecular pathways of colorectal carcinogenesis, chromosomal instability (CIN) and microsatellite instability (MSI), are considered to be mutually exclusive. Distinguishing CIN from MSI-high tumors has considerable therapeutic implications, because patients with MSI-high tumors can derive considerable benefit from immune checkpoint inhibitors, and tumors that evolved through the CIN pathway do not respond to these agents. Familial adenomatous polyposis (FAP) is a genetic syndrome that is defined by a mutation in the APC gene and is thought to lead to carcinogenesis through the CIN pathway. Here, we report a case of a young woman with FAP who was treated for medulloblastoma as a child and developed advanced MSI-high colon cancer as a young adult. Her response to second-line immunotherapy enabled resection of her colon cancer, and she is free of disease >10 months after surgery. This case highlights the potential for overlap between the CIN and MSI carcinogenic pathways and associated therapeutic implications.

A Conserved Distal Lung Regenerative Pathway in Acute Lung Injury.

Improved tools have led to a burgeoning understanding of lung regeneration in mice, but it is not yet known how these insights may be relevant to acute lung injury in humans. We report in detail two cases of fulminant idiopathic acute lung injury requiring extracorporeal membrane oxygenation in previously healthy young adults with acute respiratory distress syndrome, one of whom required lung transplantation. Biopsy specimens showed diffuse alveolar injury with a striking paucity of alveolar epithelial regeneration, rare hyaline membranes, and diffuse contiguous airspace lining by macrophages. This novel constellation was termed diffuse alveolar injury with delayed epithelization. In addition, mirroring data from murine models of lung injury/regeneration, peribronchiolar basaloid pods (previously described as squamous metaplasia) and ciliated bronchiolarization were identified in these patients and in 39% of 57 historical cases with diffuse alveolar damage. These findings demonstrate a common and clinically relevant human disease correlate for murine models of severe acute lung injury. Evidence suggests that peribronchiolar basaloid pods and bronchiolarization are related spatially and temporally and likely represent overlapping sequential stages of the response to severe distal airway injury.

Texture Analysis of Magnetic Resonance Enterography Contrast Enhancement Can Detect Fibrosis in Crohn Disease Strictures.

OBJECTIVE: The aim of the study was to investigate if texture analysis of contrast-enhanced magnetic resonance enterography (MRE) images can determine Crohn disease (CD) stricture histologic type. MATERIALS AND METHODS: A radiology report database query identified 25 pediatric patients with established CD who underwent MRE followed by bowel resection within 30 days. MRE images were reviewed to identify strictures on enteric phase T1-weighted fat-suppressed images, that were matched with sites of histologic sectioning. Regions of interest were drawn over the bowel wall and texture analysis was performed using TexRAD software (Cambridge, UK), with skewness, mean, entropy and standard deviation parameters assessed. A pathologist reviewed all stricture histology specimens to assess for active mucosal inflammation and mural fibrosis. Multivariate logistic regression and analysis of variance were performed to identify texture features associated with stricture fibrosis. RESULTS: Sixty-four bowel segments from 25 patients (mean age 16 ±â€Š2 years) with imaging-histologic correlation were included. Of note, all strictures included had undergone surgical resection with MRE imaging available within 30 days. The histologic distribution of these bowel segments included 9 segments that showed active inflammation without fibrosis, 23 segments that showed only fibrosis, and 32 mixed segments with concomitant active inflammation and fibrosis. Bivariate regression analysis demonstrated that skewness, standard deviation, entropy, and mean texture analysis features are independently associated with stricture fibrosis. Stepwise logistic regression showed that the combination of mean, skewness, and entropy texture predicted stricture fibrosis with a goodness-of-fit value of 0.995. A combination of threshold values for these 3 texture analysis parameters was able to correctly classify 100% of the strictures in the study cohort for presence (55/55) and absence (9/9) of fibrosis. CONCLUSIONS: MRE texture analysis (MRE-TA) texture features can differentiate CD stricture types and accurately detect fibrosis.

Circular RNAs are abundant, conserved, and associated with ALU repeats.

Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a "backsplice") and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.

Identification of driver genes in hepatocellular carcinoma by exome sequencing.

UNLABELLED: Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma (HCC). As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in HCC. We performed whole-exome sequencing on 87 HCCs and matched normal adjacent tissues to an average coverage of 59×. The overall mutation rate was roughly two mutations per Mb, with a median of 45 nonsynonymous mutations that altered the amino acid sequence (range, 2-381). We found recurrent mutations in several genes with high transcript levels: TP53 (18%); CTNNB1 (10%); KEAP1 (8%); C16orf62 (8%); MLL4 (7%); and RAC2 (5%). Significantly affected gene families include the nucleotide-binding domain and leucine-rich repeat-containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. CONCLUSION: The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of HCC.

Acoustic separation and concentration of exosomes for nucleotide detection: ASCENDx.

Efficient isolation and analysis of exosomal biomarkers hold transformative potential in biomedical applications. However, current methods are prone to contamination and require costly consumables, expensive equipment, and skilled personnel. Here, we introduce an innovative spaceship-like disc that allows Acoustic Separation and Concentration of Exosomes and Nucleotide Detection: ASCENDx. We created ASCENDx to use acoustically driven disc rotation on a spinning droplet to generate swift separation and concentration of exosomes from patient plasma samples. Integrated plasmonic nanostars on the ASCENDx disc enable label-free detection of enriched exosomes via surface-enhanced Raman scattering. Direct detection of circulating exosomal microRNA biomarkers from patient plasma samples by the ASCENDx platform facilitated a diagnostic assay for colorectal cancer with 95.8% sensitivity and 100% specificity. ASCENDx overcomes existing limitations in exosome-based molecular diagnostics and holds a powerful position for future biomedical research, precision medicine, and point-of-care medical diagnostics.

Natural history study of hepatic glycogen storage disease type IV and comparison to Gbe1ys/ys model.

BackgroundGlycogen storage disease type IV (GSD IV) is an ultrarare autosomal recessive disorder that causes deficiency of functional glycogen branching enzyme and formation of abnormally structured glycogen termed polyglucosan. GSD IV has traditionally been categorized based on primary hepatic or neuromuscular involvement, with hepatic GSD IV subclassified as discrete subtypes: classic (progressive) and nonprogressive.MethodsTo better understand the progression of liver disease in GSD IV, we present clinical and histopathology data from 23 patients from around the world and characterized the liver involvement in the Gbe1ys/ys knockin mouse model.ResultsWe propose an alternative to the established subtype-based terminology for characterizing liver disease in GSD IV and recognize 3 tiers of disease severity: (i) "severe progressive" liver disease, (ii) "intermediate progressive" liver disease, and (iii) "attenuated" liver disease. Analysis of liver pathology revealed that risk for liver failure cannot be predicted from liver biopsy findings alone in individuals affected by GSD IV. Moreover, analysis of postmortem liver pathology from an individual who died over 40 years after being diagnosed with nonprogressive hepatic GSD IV in childhood verified that liver fibrosis did not regress. Last, characterization of the liver involvement in a mouse model known to recapitulate the adult-onset neurodegenerative form of GSD IV (Gbe1ys/ys mouse model) demonstrated hepatic disease.ConclusionOur findings challenge the established subtype-based view of GSD IV and suggest that liver disease severity among patients with GSD IV represents a disease continuum.Trial registrationClinicalTrials.gov NCT02683512FundingNone.

Breast cancer exploits neural signaling pathways for bone-to-meninges metastasis.

The molecular mechanisms that regulate breast cancer cell (BCC) metastasis and proliferation within the leptomeninges (LM) are poorly understood, which limits the development of effective therapies. In this work, we show that BCCs in mice can invade the LM by abluminal migration along blood vessels that connect vertebral or calvarial bone marrow and meninges, bypassing the blood-brain barrier. This process is dependent on BCC engagement with vascular basement membrane laminin through expression of the neuronal pathfinding molecule integrin α6. Once in the LM, BCCs colocalize with perivascular meningeal macrophages and induce their expression of the prosurvival neurotrophin glial-derived neurotrophic factor (GDNF). Intrathecal GDNF blockade, macrophage-specific GDNF ablation, or deletion of the GDNF receptor neural cell adhesion molecule (NCAM) from BCCs inhibits breast cancer growth within the LM. These data suggest integrin α6 and the GDNF signaling axis as new therapeutic targets against breast cancer LM metastasis.

Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk.

Human genome-wide association studies have linked single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 near the INK4/ARF (CDKN2a/b) locus with susceptibility to atherosclerotic vascular disease (ASVD). Although this locus encodes three well-characterized tumor suppressors, p16(INK4a), p15(INK4b), and ARF, the SNPs most strongly associated with ASVD are ∼120 kb from the nearest coding gene within a long non-coding RNA (ncRNA) known as ANRIL (CDKN2BAS). While individuals homozygous for the atherosclerotic risk allele show decreased expression of ANRIL and the coding INK4/ARF transcripts, the mechanism by which such distant genetic variants influence INK4/ARF expression is unknown. Here, using rapid amplification of cDNA ends (RACE) and analysis of next-generation RNA sequencing datasets, we determined the structure and abundance of multiple ANRIL species. Each of these species was present at very low copy numbers in primary and cultured cells; however, only the expression of ANRIL isoforms containing exons proximal to the INK4/ARF locus correlated with the ASVD risk alleles. Surprisingly, RACE also identified transcripts containing non-colinear ANRIL exonic sequences, whose expression also correlated with genotype and INK4/ARF expression. These non-polyadenylated RNAs resisted RNAse R digestion and could be PCR amplified using outward-facing primers, suggesting they represent circular RNA structures that could arise from by-products of mRNA splicing. Next-generation DNA sequencing and splice prediction algorithms identified polymorphisms within the ASVD risk interval that may regulate ANRIL splicing and circular ANRIL (cANRIL) production. These results identify novel circular RNA products emanating from the ANRIL locus and suggest causal variants at 9p21.3 regulate INK4/ARF expression and ASVD risk by modulating ANRIL expression and/or structure.

miRNA probe integrated biosensor platform using bimetallic nanostars for amplification-free multiplexed detection of circulating colorectal cancer biomarkers in clinical samples.

There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.

BET Protein Inhibition Regulates Macrophage Chromatin Accessibility and Microbiota-Dependent Colitis.

Introduction: In colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice. Methods: We performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10-/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization. Results: Treatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo resulted in a mild reduction in colitis severity as compared with vehicle-treated mice. Conclusion: We identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis.

Delivery of therapeutic carbon monoxide by gas-entrapping materials.

Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

Nanopore Flongle Sequencing as a Rapid, Single-Specimen Clinical Test for Fusion Detection.

The identification of gene fusions is a cornerstone of diagnosis and treatment decision making for tumors of many forms and primary sites. Diverse molecular approaches are currently used for the molecular diagnosis of fusions, but few permit broad, partner agnostic detection of fusions over multiple potential targets. We previously described the combination of nanopore sequencing with the anchored multiplex PCR technique to permit a rapid testing paradigm. Recently, a new platform for nanopore sequencing has become publicly available, the Flongle flow cell from Oxford Nanopore Technologies, which offers lower throughput, but lower price testing. Here, we describe the results of retesting of 15 specimens previously tested with both Illumina and Oxford Nanopore Technologies MinION sequencing. Furthermore, we additionally blindly tested 13 specimens that had undergone clinical Illumina-based sequencing. The Flongle sequencing pipeline removed key complexities of a multiplexed nanopore sequencing protocol, reduced sequencing turnaround time, and showed excellent concordance with Illumina results. It was particularly strong in identifying notoriously difficult to detect CIC-DUX4 translocations. The Flongle sequencing pipeline may be the assay of choice for deployment in small- to medium-sized molecular laboratories.

Primary hepatic neoplasms arising in cirrhotic livers can have a variable spectrum of neuroendocrine differentiation.

Primary hepatic neoplasms with neuroendocrine differentiation are extremely rare. Their clinicopathological features and molecular genetic basis are largely unknown. We identified four cases of primary hepatic neoplasms with neuroendocrine differentiation. Electronic medical records were reviewed for clinical history, imaging findings, laboratory results, and follow-up. Pathology slides, immunohistochemistry, and ancillary studies were reviewed. There were two females and two males with age ranging from 52 to 74 years. There was one amphicrine carcinoma with tumor cells simultaneously demonstrating both hepatocellular and neuroendocrine differentiation, one mixed hepatocellular-neuroendocrine carcinoma (NEC) with hepatocellular component intermingled with neuroendocrine component, one small cell NEC, and one well-differentiated neuroendocrine tumor. Next- generation sequencing of the mixed hepatocellular-NEC and small cell NEC showed molecular/genetic alterations commonly seen in hepatocellular carcinoma (HCC). All four cases arose in a background of cirrhosis. Primary hepatic neoplasms arising in cirrhotic livers can have a spectrum of neuroendocrine differentiation. Presence of a NEC component may be an indicator of aggressiveness. In addition, primary hepatic carcinomas with neuroendocrine differentiation likely share the same molecular pathways as HCC.

Personalized Radiation Attenuating Materials for Gastrointestinal Mucosal Protection.

Cancer patients undergoing therapeutic radiation routinely develop injury of the adjacent gastrointestinal (GI) tract mucosa due to treatment. To reduce radiation dose to critical GI structures including the rectum and oral mucosa, 3D-printed GI radioprotective devices composed of high-Z materials are generated from patient CT scans. In a radiation proctitis rat model, a significant reduction in crypt injury is demonstrated with the device compared to without (p < 0.0087). Optimal device placement for radiation attenuation is further confirmed in a swine model. Dosimetric modeling in oral cavity cancer patients demonstrates a 30% radiation dose reduction to the normal buccal mucosa and a 15.2% dose reduction in the rectum for prostate cancer patients with the radioprotectant material in place compared to without. Finally, it is found that the rectal radioprotectant device is more cost-effective compared to a hydrogel rectal spacer. Taken together, these data suggest that personalized radioprotectant devices may be used to reduce GI tissue injury in cancer patients undergoing therapeutic radiation.

LGR5 in Barrett's Esophagus and its Utility in Predicting Patients at Increased Risk of Advanced Neoplasia.

INTRODUCTION: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia. METHODS: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors. The LGR5 stain was evaluated using 1 qualitative and 2 quantitative parameters, using manual and automated platforms. RESULTS: Surface LGR5 expression was mainly seen in high-grade dysplasia (12/18) compared with low-grade dysplasia (1/8) and nondysplastic BE (0/17) (P < 0.0001). In contrast to nondysplastic BE, low- and high-grade dysplasia showed a higher percentage of mean number of LGR5-positive crypts per patient (P < 0.0001) and an increase in the mean number of LGR5 transcripts per cell (P < 0.0001). The mean percentage of LGR5-positive crypts per patient and the mean number of LGR5 transcripts per cell were also significantly higher in nondysplastic BE from progressor compared with nonprogressor (P < 0.0001, P = 0.014). The sensitivity and specificity of LGR5 for distinguishing progressor from nonprogressor were 50% and 87%, respectively. DISCUSSION: BE-related advanced neoplasia shows an expansion of the LGR5-positive cellular compartment, supporting its role as a stem cell marker in this disease. Quantitative LGR5 expression and surface epithelial reactivity are novel biomarkers of increased risk of progression to advanced neoplasia in BE.