WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

Assessment of doxorubicin-induced remodeling of Ca2+ signaling and associated Ca2+ regulating proteins in MDA-MB-231 breast cancer cells.

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 µM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 µM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.

High-Throughput Fluorescence Assays for Ion Channels and GPCRs.

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.

A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase.

We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten constant (Km ) of 15.89 µm. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analogue. In vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analogue.

MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.

The dose of omeprazole required to achieve adequate intraesophageal acid suppression in patients with gastroesophageal junction specialized intestinal metaplasia and Barrett's esophagus.

BACKGROUND: The mainstay of medical therapy for Barrett's esophagus is normalization of esophageal acid exposure with proton pump inhibitors (PPIs). However, the optimal dose and whether once daily or twice daily is required for acid suppression is unknown. AIM: The purpose of this study was to assess whether adequate intra-esophageal acid suppression could be achieved with once daily versus twice daily omeprazole in patients with gastroesophageal specialized intestinal metaplasia (GEJSIM), short-segment (SSBE) and long-segment Barrett's esophagus (LSBE). METHODS: Patients with GEJSIM and Barrett's esophagus underwent upper endoscopy with 48-h wireless pH capsule while on once daily 20 mg omeprazole for at least 1 week. If intra-esophageal acid was not adequately controlled, defined as pH value <4 for greater than 4.2 % of the time during the second 24-h period, omeprazole was increased to twice daily for 1 week and upper endoscopy with wireless pH capsule was repeated. RESULTS: A total of 36 patients completed the study (10 patients had GEJSIM, 16 patients had SSBE, and 10 patients had LSBE). Normalization of intraesophageal pH was achieved in 28 patients (78 %) with once daily PPI and eight patients required twice daily PPI. There was no significant difference between the three groups in the proportion of patients requiring high dose PPI (GEJSIM 10 %, SSBE 25 %, LSBE 30 %, p = 0.526). CONCLUSIONS: The majority of patients with Barrett's esophagus were controlled with once daily low dose PPI and only a minority required twice daily dosing, regardless of the length of Barrett's mucosa.

Reducing Metabolic Bone Disease Burden in Intestinal Failure Children on Home Parenteral Nutrition.

Objective: To determine the prevalence of secondary hyperparathyroidism in a cohort of pediatric patients receiving home parenteral nutrition. Methods: For a service review, a population-based cohort of 37 pediatric intestinal failure patients receiving long-term parenteral nutrition that underwent serial biochemical monitoring during a study period of approximately 4 years were examined. Following the production of an algorithm, a follow-up audit was carried out (n = 33) after approximately 6 months. Results: Of the 37 patients examined in the initial service review, 22 (59%) were found to have an elevated parathyroid hormone (PTH) during the period of monitoring and 5 (14%) had a persistently elevated PTH. In the follow-up audit following the implementation of an algorithm, the number with elevated PTH reduced to 6 (18%) and no patients had persistently high levels. Conclusion: Elevated PTH is a common biochemical finding in pediatric intestinal failure patients receiving home parenteral nutrition and its presence should alert clinicians to the need to optimize nutritional parameters such as calcium to phosphate molar ratio and vitamin D status; failure to do so may increase the future burden of metabolic bone disease in such patients. We propose that an algorithm may help in this endeavor.

Bcl6 is a subset-defining transcription factor of lymphoid tissue inducer-like ILC3.

Innate lymphoid cells (ILCs) are tissue-resident effector cells with roles in tissue homeostasis, protective immunity, and inflammatory disease. Group 3 ILCs (ILC3s) are classically defined by the master transcription factor RORγt. However, ILC3 can be further subdivided into subsets that share type 3 effector modules that exhibit significant ontological, transcriptional, phenotypic, and functional heterogeneity. Notably lymphoid tissue inducer (LTi)-like ILC3s mediate effector functions not typically associated with other RORγt-expressing lymphocytes, suggesting that additional transcription factors contribute to dictate ILC3 subset phenotypes. Here, we identify Bcl6 as a subset-defining transcription factor of LTi-like ILC3s in mice and humans. Deletion of Bcl6 results in dysregulation of the LTi-like ILC3 transcriptional program and markedly enhances expression of interleukin-17A (IL-17A) and IL-17F in LTi-like ILC3s in a manner in part dependent upon the commensal microbiota-and associated with worsened inflammation in a model of colitis. Together, these findings redefine our understanding of ILC3 subset biology.

MITF Is Regulated by Redox Signals Controlled by the Selenoprotein Thioredoxin Reductase 1.

TR1 and other selenoproteins have paradoxical effects in melanocytes and melanomas. Increasing selenoprotein activity with supplemental selenium in a mouse model of UV-induced melanoma prevents oxidative damage to melanocytes and delays melanoma tumor formation. However, TR1 itself is positively associated with progression in human melanomas and facilitates metastasis in melanoma xenografts. Here, we report that melanocytes expressing a microRNA directed against TR1 (TR1low) grow more slowly than control cell lines and contain significantly less melanin. This phenotype is associated with lower tyrosinase (TYR) activity and reduced transcription of tyrosinase-like protein-1 (TYRP1). Melanoma cells in which the TR1 gene (TXNRD1) was disrupted using Crispr/Cas9 showed more dramatic effects including the complete loss of the melanocyte-specific isoform of MITF; other MITF isoforms were unaffected. We provide evidence that TR1 depletion results in oxidation of MITF itself. This newly discovered mechanism for redox modification of MITF has profound implications for controlling both pigmentation and tumorigenesis in cells of the melanocyte lineage.

Can quality of care for patients with cirrhosis be measured?

INTRODUCTION: The ultimate purpose of measuring quality of care is to discriminate between healthcare providers in order to motivate improvement. Recently, a set of evidence-based indicators has been proposed for measurement of processes of care for patients with cirrhosis, for example early endoscopy for variceal bleeding. The objective of this study was to determine whether these indicators can be measured in a reliable and automated fashion in routine practice. MATERIALS AND METHODS: We applied the top five indicators, based on agreement of a panel of experts, to hospitalized adults at our institution over a 3-year period. RESULTS: Only two of the indicators could be reliably measured on the basis of the published wording, and these two still required physician chart review. After applying some assumptions, the indicators were met in 46-100% of cases. None of the indicators was linked to a single physician or institution in all cases, and none occurred with sufficient frequency to discriminate quality between providers. CONCLUSION: Measuring quality of care in cirrhosis is a laudable objective, but current indicators are not yet ready for administrative use.

Esophageal motor disorders in adults with eosinophilic esophagitis.

BACKGROUND: An association between eosinophilic esophagitis (EoE) and esophageal motility disorders has been described in small studies. AIMS: The aim of this study was to describe the prevalence of esophageal motor disorders in a large cohort of adults with EoE and examine whether an association exists between esophageal dysmotility and dysphagia. METHODS: A retrospective review of esophageal manometry studies in adult EoE patients was performed. Tracings were reviewed for abnormalities including nutcracker esophagus and ineffective swallows, defined as low amplitude peristalsis (<30 mmHg) or non-propagating contractions. Ineffective esophageal motility (IEM) was categorized as mild (30-40% ineffective swallows), moderate (50-60% ineffective swallows), and severe (≥70% ineffective swallows). Dysphagia was graded on a 0-3 scale for frequency and severity. RESULTS: Seventy-five tracings from EoE patients were reviewed (85% male, mean age 41 ± 12 years). IEM was identified in 25 patients and categorized as mild (n = 13), moderate (n = 6), and severe (n = 6). Nutcracker esophagus was found in three patients. There was no significant difference in eosinophil count among the motility groups: normal 46.5 ± 3.1, mild IEM 56.9 ± 36.9, moderate IEM 45.5 ± 23.7, severe IEM 34.3 ± 12.6 (P = 0.157). CONCLUSIONS: In this cohort of EoE patients, the majority had normal esophageal motility studies, although a subset of these patients had some esophageal dysmotility. It is unlikely that esophageal dysmotility is a major contributing factor to dysphagia, although it is reasonable to consider esophageal manometry testing in EoE patients to identify potential abnormalities of the smooth muscle esophagus.

Exercise-associated hyponatremia: the influence of pre-exercise carbohydrate status combined with high volume fluid intake on sodium concentrations and fluid balance.

To evaluate the effect of hydration and carbohydrate (CHO) status on plasma sodium, fluid balance, and regulatory factors (IL-6 & ADH) during and after exercise; 10 males completed the following conditions: low CHO, euhydrated (fluid intake = sweat loss) (LCEH); low CHO, dehydrated (no fluid) (LCDH); high CHO, euhydrated (HCEH); and high CHO, dehydrated (HCDH). Each trial consisted of 90-min cycling at 60% VO(2) max in a 35°C environment followed by 3-h rehydration (RH). During RH, subjects received either 150% of sweat loss (LCDH & HCDH) or an additional 50% of sweat loss (LCEH and HCEH). Blood was analyzed for glucose, IL-6, ADH, and Na(+). Post-exercise Na(+) was greater (p < 0.001) for LCDH and HCDH (141.7 + 0.72 and 141.6 + 0.4 mM) versus LCEH and HCEH (136.4 + 0.6 and 135.9 + 0.3 mM). Post-exercise IL-6 was similar in all conditions, and post-exercise ADH was greater (p = 0.01) in dehydrated versus euhydrated conditions. The rate of urine production was greater in HCEH (7.59 + 3.0 mL/min) compared to all other conditions (3.86 + 2.2, 5.29 + 3.1, and 2.96 + 1.1 mL/min for LCDH, LCEH, and HCDH, respectively). Despite CHO and hydration manipulations, no regulatory effects of IL-6 and ADH on plasma [Na(+)] were observed. With euhydration during exercise and additional fluid consumed during recovery, a high-CHO status increased urinary output during recovery, and it decreased the frequency of hyponatremia (Na(+) < 135 mM). Therefore, a high-CHO status may provide some protection against exercise-associated hyponatremia.

Possible COVID-19 reinfection in a patient with X-linked agammaglobulinaemia.

This report highlights the case of a patient with X-linked agammaglobulinaemia (XLA) and resultant bronchiectasis who was discharged from hospital after recovering from real-time reverse transcriptase-PCR positive COVID-19 infection having had a subsequent negative swab and resolution of symptoms, but was readmitted 3 weeks later with recrudescent symptoms and a further positive swab. Although there are reports of COVID-19 infection in XLA, for the first time we report a case of possible reinfection. Lessons learnt from this case include the potential for reinfection of COVID-19 in a patient with a weakened immune system and the importance of repeating COVID-19 swabs in inpatients. Extra caution needs to be taken when providing care in groups of patients who have a weakened or absent immune system.

An ARHGAP25 variant links aberrant Rac1 function to early-onset skeletal fragility.

Ras homologous guanosine triphosphatases (RhoGTPases) control several cellular functions, including cytoskeletal actin remodeling and cell migration. Their activities are downregulated by GTPase-activating proteins (GAPs). Although RhoGTPases are implicated in bone remodeling and osteoclast and osteoblast function, their significance in human bone health and disease remains elusive. Here, we report defective RhoGTPase regulation as a cause of severe, early-onset, autosomal-dominant skeletal fragility in a three-generation Finnish family. Affected individuals (n = 13) presented with multiple low-energy peripheral and vertebral fractures despite normal bone mineral density (BMD). Bone histomorphometry suggested reduced bone volume, low surface area covered by osteoblasts and osteoclasts, and low bone turnover. Exome sequencing identified a novel heterozygous missense variant c.652G>A (p.G218R) in ARHGAP25, encoding a GAP for Rho-family GTPase Rac1. Variants in the ARHGAP25 5' untranslated region (UTR) also associated with BMD and fracture risk in the general population, across multiple genomewide association study (GWAS) meta-analyses (lead variant rs10048745). ARHGAP25 messenger RNA (mRNA) was expressed in macrophage colony-stimulating factor (M-CSF)-stimulated human monocytes and mouse osteoblasts, indicating a possible role for ARHGAP25 in osteoclast and osteoblast differentiation and activity. Studies on subject-derived osteoclasts from peripheral blood mononuclear cells did not reveal robust defects in mature osteoclast formation or resorptive activity. However, analysis of osteosarcoma cells overexpressing the ARHGAP25 G218R-mutant, combined with structural modeling, confirmed that the mutant protein had decreased GAP-activity against Rac1, resulting in elevated Rac1 activity, increased cell spreading, and membrane ruffling. Our findings indicate that mutated ARHGAP25 causes aberrant Rac1 function and consequently abnormal bone metabolism, highlighting the importance of RhoGAP signaling in bone metabolism in familial forms of skeletal fragility and in the general population, and expanding our understanding of the molecular pathways underlying skeletal fragility. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

COVID-19 presenting as fulminant hepatic failure: A case report.

INTRODUCTION: Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) responsible for the COVID-19 pandemic has spread from Wuhan, China in December, 2019 to 216 countries and territories as of September 10, 2020 with 27.74 million cases and 899,911 confirmed deaths. The spectrum of disease is most commonly seen as a viral pneumonia with high grade fevers, shortness of breath, dry cough, and chest pain with radiologic evidence of bilateral, interstitial, ground glass opacities, and peripheral lung consolidation. Liver chemistries are frequently abnormal, with transaminases shown to be one-two times the upper limit of normal in most instances. The full spectrum of gastrointestinal involvement of the SARS-CoV-2 infection has yet to be fully seen.Patient concerns: We present a case of a young woman with SLE who developed severe abdominal pain, nausea and vomiting, rapidly progressing to acute hepatic failure and tested positive for SARS-CoV-2 infection. She had no respiratory symptoms. DIAGNOSIS: A thorough work-up of acute liver failure including liver biopsy confirmed acute hepatitis with viral like changes. Common viral causes of liver failure were ruled out. The patient had no recent travel history. INTERVENTIONS: The patient was started on hydroxychloroquine due to SLE, treated with N-Acetyl-Cysteine, and methylprednisolone. OUTCOMES: The patient improved with resolution of encephalopathy and normalization of her liver chemistries without any development of respiratory illness. CONCLUSION: This case details a unique presentation of likely SARS-CoV-2 infection. Until now, the literature has primarily described a respiratory illness and liver injury with mild transaminase elevations. Significant liver injury progressing to acute liver failure should be considered in those with SARS-CoV-2 infection.

Acute liver failure in a military recruit treated with valproic acid and harboring a previously unrecognized POLG-1 mutation.

Patients with mutations in the POLG-1 gene often are afflicted with drug-resistant seizures at an early age and have an increased risk of valproic acid-induced acute liver failure. Severe valproate hepatotoxicity most commonly arises in children within the first 3 months of treatment with an overall estimated incidence of 1 in 40,000 treated patients. Due to high mortality rates among transplanted children, many experts consider valproic acid-induced acute liver failure in patients with mitochondrial disorders to be a contraindication to liver transplant. We report the successful use of liver transplantation in a young man with valproic acid-associated acute liver failure harboring a previously unrecognized POLG-1 mutation.

Colonic perforation during screening CT colonography using automated CO2 insufflation in an asymptomatic adult.

CT colonography has become a potential alternative technique to optical colonoscopy for the detection of colorectal polyps and cancer. While considered safer than optical colonoscopy, CT colonography is not without risk. We report a case of colonic perforation during CT colonography using automated CO(2) insufflation and present procedural changes to help minimize the adverse effects of perforation when it occurs.

The effect of visual and auditory cues on seat preference in an opera theater.

Opera performance conveys both visual and auditory information to an audience, and so opera theaters should be evaluated in both domains. This study investigates the effect of static visual and auditory cues on seat preference in an opera theater. Acoustical parameters were measured and visibility was analyzed for nine seats. Subjective assessments for visual-only, auditory-only, and auditory-visual preferences for these seat positions were made through paired-comparison tests. In the cases of visual-only and auditory-only subjective evaluations, preference judgment tests on a rating scale were also employed. Visual stimuli were based on still photographs, and auditory stimuli were based on binaural impulse responses convolved with a solo tenor recording. For the visual-only experiment, preference is predicted well by measurements taken related to the angle of seats from the theater midline at the center of the stage, the size of the photographed stage view, the visual obstruction, and the distance from the stage. Sound pressure level was the dominant predictor of auditory preference in the auditory-only experiment. In the cross-modal experiments, both auditory and visual preferences were shown to contribute to overall impression, but auditory cues were more influential than the static visual cues. The results show that both a positive visual-only or a positive auditory-only evaluations positively contribute to the assessments of seat quality.