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PURPOSE: Otitis media is the most common condition diagnosed by pediatricians and is estimated to affect approximately 70% of the pediatric population. The goal of this study was to evaluate the effectiveness of otoscopy and multifrequency tympanometry (MFT) for diagnosis of otitis media in children. METHOD: Twenty-one children, age 1 to 10 years, who were seeking medical treatment for suspected middle ear disease were selected to participate. Data were collected prior to myringotomy to determine the sensitivity and specificity rates of the following otologic and audiologic measures: (a) pneumatic otoscopy, (b) conventional tympanometry, and (c) MFT. For this study, the 'gold standard,' myringotomy, was used along with pneumatic otoscopy to determine the effectiveness, sensitivity, and specificity of conventional 226-Hz tympanometry, 678-Hz tympanometry, and 1000-Hz tympanometry to predict middle ear disease. RESULTS: The diagnoses provided with pneumatic otoscopy and tympanometry were both similar, agreeing in diagnosis 80%-100% of the time. The diagnoses from 678-Hz and 1000-Hz tympanometry were nearly equal and proved to detect abnormality at a higher rate. CONCLUSIONS: MFT is recommended on a routine basis with children having a history of otitis media, or else abnormal or notched 226-Hz tympanograms. Further research with a larger sample size will illuminate the possible predictive potential of MFT in otitis media. [ABSTRACT FROM AUTHOR]

Purpose: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSIs) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood., Experimental Design: We applied targeted cell-free DNA sequencing to 126 mCRPC patients from three academic cancer centers, and separately performed genome-wide cell-free DNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome-positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 mCRPC patients, respectively, to develop and validate a stem-like signature, which we inferred from cell-free DNA., Results: Targeted cell-free DNA sequencing detected AR/enhancer alterations prior to first-line ARSIs which correlated with significantly worse PFS (p = 0.01; HR = 2.12) and OS (p = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (p < 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cell-free DNA revealed enrichment for stemness-associated transcription factors in lethal mCRPC patients. The resulting stemness signature was then validated in a completely held-out cohort of 80 mCRPC patients profiled by tumor RNA sequencing., Conclusions: We analyzed a total of 220 mCRPC patients, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness.

Purpose: Our goal was to demonstrate that lymphatic drainage fluid (lymph) has improved sensitivity in quantifying postoperative minimal residual disease (MRD) in locally advanced human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) compared with plasma, and leverage this novel biofluid for patient risk stratification., Experimental Design: We prospectively collected lymph samples from neck drains of 106 patients with HPV (+) OPSCC, along with 67 matched plasma samples, 24 hours after surgery. PCR and next-generation sequencing were used to quantify cancer-associated cell-free HPV (cf-HPV) and tumor-informed variants in lymph and plasma. Next, lymph cf-HPV and variants were compared with TNM stage, extranodal extension (ENE), and composite definitions of high-risk pathology. We then created a machine learning model, informed by lymph MRD and clinicopathologic features, to compare with progression-free survival (PFS)., Results: Postoperative lymph was enriched with cf-HPV compared with plasma (P < 0.0001) and correlated with pN2 stage (P = 0.003), ENE (P < 0.0001), and trial-defined pathologic risk criteria (mean AUC = 0.78). In addition, the lymph mutation number and variant allele frequency were higher in pN2 ENE (+) necks than in pN1 ENE (+) (P = 0.03, P = 0.02) or pN0-N1 ENE (-) (P = 0.04, P = 0.03, respectively). The lymph MRD-informed risk model demonstrated inferior PFS in high-risk patients (AUC = 0.96, P < 0.0001)., Conclusions: Variant and cf-HPV quantification, performed in 24-hour postoperative lymph samples, reflects single- and multifeature high-risk pathologic criteria. Incorporating lymphatic MRD and clinicopathologic feature analysis can stratify PFS early after surgery in patients with HPV (+) head and neck cancer. See related commentary by Shannon and Iyer, p. 1223., (©2023 The Authors; Published by the American Association for Cancer Research.)

Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n = 25). We then combined these samples with surgical resection (n = 6) and publicly available samples to increase statistical power (n = 80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness, and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n = 268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens, including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making., (© 2023. Nature Publishing Group UK.)

Motivation: Detection of genomic alterations in circulating tumor DNA (ctDNA) is currently used for active clinical monitoring of cancer progression and treatment response. While methods for analysis of small mutations are more developed, strategies for detecting structural variants (SVs) in ctDNA are limited. Additionally, reproducibly calling small-scale mutations, copy number alterations, and SVs in ctDNA is challenging due to the lack to unified tools for these different classes of variants., Results: We developed a unified pipeline for the analysis of ctDNA [Pipeline for the Analysis of ctDNA (PACT)] that accurately detects SVs and consistently outperformed similar tools when applied to simulated, cell line, and clinical data. We provide PACT in the form of a Common Workflow Language pipeline which can be run by popular workflow management systems in high-performance computing environments., Availability and Implementation: PACT is freely available at https://github.com/ChrisMaherLab/PACT., (© The Author(s) 2023. Published by Oxford University Press.)

Traditional cold chain systems of collection, transportation, and storage of biofluid specimens for eventual analysis pose a huge financial and environmental burden. These systems are impractical in pre-hospital and resource-limited settings, where refrigeration and electricity are not reliable or even available. Here, we develop an innovative technology using metal-organic frameworks (MOFs), a novel class of organic-inorganic hybrids with high thermal stability, as encapsulates for preserving the integrity of protein biomarkers in biofluids under ambient or non-refrigerated storage conditions. We encapsulate prostate-specific antigen (PSA) in whole patient plasma using hydrophilic zeolitic imidazolate framework-90 (ZIF-90) for preservation at 40 °C for 4 weeks and eventual on-demand reconstitution for antibody-based assays with recovery above 95% compared to storage at -20 °C. Without ZIF-90 encapsulation, only 10-30% of the PSA immunoactivity remained. Furthermore, we demonstrate encapsulation of multiple cancer biomarker proteins in whole patient plasma using ZIF-8 or ZIF-90 encapsulants for eventual on-demand reconstitution and analysis after 1 week at 40 °C. Overall, MOF encapsulation of patient biofluids is important as climate change may be affecting the stability and increase costs of maintaining biospecimen cold chain custody for the collection, transportation, and storage of biospecimens prior to analysis or for biobanking regardless of any countries' affluence.

Circulating tumor DNA (ctDNA) sensitivity remains subpar for molecular residual disease (MRD) detection in bladder cancer patients. To remedy this problem, we focused on the biofluid most proximal to the disease, urine, and analyzed urine tumor DNA in 74 localized bladder cancer patients. We integrated ultra-low-pass whole genome sequencing (ULP-WGS) with urine cancer personalized profiling by deep sequencing (uCAPP-Seq) to achieve sensitive MRD detection and predict overall survival. Variant allele frequency, inferred tumor mutational burden, and copy number-derived tumor fraction levels in urine cell-free DNA (cfDNA) significantly predicted pathologic complete response status, far better than plasma ctDNA was able to. A random forest model incorporating these urine cfDNA-derived factors with leave-one-out cross-validation was 87% sensitive for predicting residual disease in reference to gold-standard surgical pathology. Both progression-free survival (HR = 3.00, p = 0.01) and overall survival (HR = 4.81, p = 0.009) were dramatically worse by Kaplan-Meier analysis for patients predicted by the model to have MRD, which was corroborated by Cox regression analysis. Additional survival analyses performed on muscle-invasive, neoadjuvant chemotherapy, and held-out validation subgroups corroborated these findings. In summary, we profiled urine samples from 74 patients with localized bladder cancer and used urine cfDNA multi-omics to detect MRD sensitively and predict survival accurately., (© 2023. The Author(s).)

Purpose: Low-dose computed tomography (LDCT) screening of high-risk patients decreases lung cancer-related mortality. However, high false-positive rates associated with LDCT result in unnecessary interventions. To distinguish non-small-cell lung cancer (NSCLC) from benign nodules, in the present study, we integrated cellular liquid biomarkers in patients with suspicious lung nodules (lung cancer screening reporting and data system [Lung-RADS] 4)., Methods: Prospectively, 7.5 mL of blood was collected from 221 individuals (training set: 90 nonscreened NSCLC patients, 74 high-risk screening patients with no/benign nodules [Lung-RADS 1-3], and 20 never smokers; validation set: 37 patients with suspicious nodules [Lung-RADS 4]). Circulating tumor cells (CTCs), CTC clusters, and tumor-macrophage fusion (TMF) cells were identified by blinded analyses. Screening patients underwent a median of two LDCTs (range, 1-4) with a median surveillance time of 30 (range, 11-50) months., Results: In the validation set of 37 Lung-RADS 4 patients, all circulating cellular biomarker counts ( P < .005; Wilcoxon test) and positivity rates were significantly higher in 23 biopsy-proven NSCLC patients (CTCs: 23 of 23 [100%], CTC clusters: 6 of 23 [26.1%], and TMF cells: 15 of 23 [65.2%]) than in 14 patients with biopsy-proven benign nodules (6 of 14 [42.9%], 0 of 14 [0%], and 2 of 14 [14.3%]). On the basis of cutoff values from the training set, logistic regression with receiver operating characteristic and area under the curve analyses demonstrated that CTCs (sensitivity: 0.870, specificity: 1.0, and area under the curve: 0.989) and TMF cells (0.652; 0.880; 0.790) complement LDCT in diagnosing NSCLC in Lung-RADS 4 patients., Conclusion: Cellular liquid biomarkers have a potential to complement LDCT interpretation of suspicious Lung-RADS 4 nodules to distinguish NSCLC from benign lung nodules. A future prospective, large-scale, multicenter clinical trial should validate the role of cellular liquid biomarkers in improving diagnostic accuracy in high-risk patients with Lung-RADS 4 nodules., Competing Interests: Kevin F. Staveley-O'CarrollHonoraria: AstraZeneca Klaus PantelHonoraria: Agena Bioscience, Novartis, Roche, Medac, Impulze, Ipsen, Sanofi, Merck KGaA, MSD, Beiersdorf, Galderma, Hummingbird, Illumina, Hello Healthcare, Menarini Silicon Biosystems, Abcam, Atheneum, CureVac, DeciBio, Inflection Biosciences, Molecular Health, TacticsConsulting or Advisory Role: Sanofi, Agena Bioscience, Hummingbird Diagnostics, Menarini Silicon BiosystemsResearch Funding: Janssen Diagnostics, Cancer-ID (Inst)Patents, Royalties, Other Intellectual Property: Application No. WO2016 128125A1, application No. 17157020.3—1405, application No. 10004180.5, application No. 07825055.2, application No. 95108760.7Travel, Accommodations, Expenses: Agena Bioscience Guangfu LiPatents, Royalties, Other Intellectual Property: Ceramide nanoliposomes as a method and device for immunotherapy (Inst) Aadel A. ChaudhuriLeadership: Droplet BiosciencesStock and Other Ownership Interests: Geneoscopy, Droplet BiosciencesHonoraria: RocheConsulting or Advisory Role: Geneoscopy, Roche, Tempus, AstraZeneca/Daiichi SankyoPatents, Royalties, Other Intellectual Property: US Patent No. US8685727B2Travel, Accommodations, Expenses: Roche, Foundation MedicineOther Relationship: Roche Jussuf T. KaifiPatents, Royalties, Other Intellectual Property: Cancer Biomarker detectionNo other potential conflicts of interest were reported.

Severe immune-related adverse events (irAEs) occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). However, it is unknown whether a common baseline immunological state precedes irAE development. Here we applied mass cytometry by time of flight, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing and bulk T cell receptor (TCR) sequencing to study peripheral blood samples from patients with melanoma treated with anti-PD-1 monotherapy or anti-PD-1 and anti-CTLA-4 combination ICIs. By analyzing 93 pre- and early on-ICI blood samples and 3 patient cohorts (n = 27, 26 and 18), we found that 2 pretreatment factors in circulation-activated CD4 memory T cell abundance and TCR diversity-are associated with severe irAE development regardless of organ system involvement. We also explored on-treatment changes in TCR clonality among patients receiving combination therapy and linked our findings to the severity and timing of irAE onset. These results demonstrate circulating T cell characteristics associated with ICI-induced toxicity, with implications for improved diagnostics and clinical management., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The detection of circulating tumor DNA via liquid biopsy has become an important diagnostic test for patients with cancer. While certain commercial liquid biopsy platforms designed to detect circulating tumor DNA have been approved to guide clinical decisions in advanced solid tumors, the clinical utility of these assays for detecting minimal residual disease after curative-intent treatment of nonmetastatic disease is currently limited. Predicting disease response and relapse has considerable potential for increasing the effective implementation of neoadjuvant and adjuvant therapies. As a result, many companies are rapidly investing in the development of liquid biopsy platforms to detect circulating tumor DNA in the minimal residual disease setting. In this review, we discuss the development and clinical implementation of commercial liquid biopsy platforms for circulating tumor DNA minimal residual disease detection of solid tumors. Here, we aim to highlight the technological features that enable highly sensitive detection of tumor-derived genomic alterations, the factors that differentiate these commercial platforms, and the ongoing trials that seek to increase clinical implementation of liquid biopsies using circulating tumor DNA-based minimal residual disease detection., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors., Methods and Findings: This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort., Conclusions: Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.A.C. has patent filings related to cancer biomarkers, and has served as a consultant/advisor to Roche, Tempus, Geneoscopy, NuProbe, Daiichi Sankyo, AstraZeneca, Fenix Group International and Guidepoint. A.A.C. has stock options in Geneoscopy, research support from Roche, and ownership interests in Droplet Biosciences. A.C.H. has served on advisory boards for AstraZenica and Springworks Therapeutics. F.Q. has stocks: Centene. No potential conflicts of interest were disclosed by the other authors.

Background: The standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis., Methods and Findings: We applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4-38.9; p = 0.02) compared to utDNA MRD-negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point., Conclusions: utDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.A.C. has patent filings related to cancer biomarkers, and has served as a consultant/advisor to Roche, Tempus, Geneoscopy, NuProbe, Daiichi Sankyo, AstraZeneca, Fenix Group International and Guidepoint; A.A.C. has stock options in Geneoscopy, research support from Roche, and ownership interests in Droplet Biosciences. V.K.A. has received research funding from ORIC Pharmaceuticals and currently serves as an employee of Bristol Myers Squibb; V.K.A. has stock options in both companies. Z.L.S. serves as a consultant/advisor for Photocure, outside of the submitted work. B.C.B. discloses honoraria from Mevion Medical Systems and consulting work for Regeneron/Sanofi, outside of the submitted work. R.K.B. is employed as a hospital medicine physician for SSM Health St. Louis. F.Q. has stock options in Centene, Gilead, and Horizon Therapeutics. No potential conflicts of interest were disclosed by the other authors.

We hypothesized that circulating tumor DNA (ctDNA) molecular residual disease (MRD) analysis without prior mutational knowledge could be performed after neoadjuvant chemotherapy to assess oligometastatic colorectal cancer (CRC) treated surgically with curative intent. We also investigated urine as an alternative analyte for ctDNA MRD detection in this nongenitourinary setting., Patients and Methods: We applied AVENIO targeted next-generation sequencing to plasma, tumor, and urine samples acquired on the day of curative-intent surgery from 24 prospectively enrolled patients with oligometastatic CRC. Age-related clonal hematopoiesis was accounted for by removing variants also present in white blood cells. Plasma and urine ctDNA MRD were correlated with tumor cells detected in the surgical specimen, and adjuvant treatment strategies were proposed based on ctDNA-inferred tumor mutational burden (iTMB) and targetable alterations., Results: Seventy-one percent of patients were treated with neoadjuvant chemotherapy. Tumor-naive plasma ctDNA analysis detected MRD at a median level of 0.62% with 95% sensitivity and 100% specificity, and 94% and 77% sensitivity when only considering patients treated with neoadjuvant chemotherapy and putative driver mutations, respectively. In urine, ctDNA MRD detection specificity remained high at 100%, but sensitivity decreased to 64% with median levels being 11-fold lower than in plasma ( P < .0001). Personalized ctDNA MRD oncogenomic analysis revealed 81% of patients might have been candidates for adjuvant immunotherapy based on high iTMB or targeted therapy based on actionable PIK3CA mutations., Conclusion: Tumor-naive plasma ctDNA analysis can sensitively and specifically detect MRD in patients with oligometastatic CRC after neoadjuvant chemotherapy. Urine-based ctDNA MRD detection is also feasible; however, it is less sensitive than plasma because of significantly lower levels. Oligometastatic patients with detectable MRD may benefit from additional personalized treatment based on ctDNA-derived oncogenomic profiling., (© 2021 by American Society of Clinical Oncology.)

Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Aadel A. ChaudhuriStock and Other Ownership Interests: Geneoscopy Honoraria: Foundation Medicine, Roche Consulting or Advisory Role: Geneoscopy, Roche, Fenix Group International, Tempus Patents, Royalties, Other Intellectual Property: US Patent No. US8685727B2 Travel, Accommodations, Expenses: Roche, Foundation Medicine Other Relationship: RocheVivek K. AroraStock and Other Ownership Interests: ORIC Pharmaceuticals Consulting or Advisory Role: H3 Biomedicine, Bristol Myers Squibb, Seattle Genetics/Astellas Research Funding: ORIC Pharmaceuticals Travel, Accommodations, Expenses: H3 Biomedicine, Bristol Myers Squibb No other potential conflicts of interest were reported.

Purpose: Cell-free DNA (cfDNA) and circulating tumor cell (CTC) based liquid biopsies have emerged as potential tools to predict responses to androgen receptor (AR)-directed therapy in metastatic prostate cancer. However, due to complex mechanisms and incomplete understanding of genomic events involved in metastatic prostate cancer resistance, current assays (e.g. CTC AR-V7) demonstrate low sensitivity and remain underutilized. The recent discovery of AR enhancer amplification in >80% of metastatic patients and its association with disease resistance presents an opportunity to improve upon current assays. We hypothesized that tracking AR /enhancer genomic alterations in plasma cfDNA would detect resistance with high sensitivity and specificity., Methods: We developed a targeted sequencing and analysis method as part of a new assay called Enhancer and neighboring loci of Androgen Receptor Sequencing (EnhanceAR-Seq). We applied EnhanceAR-Seq to plasma collected from 40 patients with metastatic prostate cancer treated with AR-directed therapy to monitor AR /enhancer genomic alterations and correlate these events with therapy resistance, progression-free survival (PFS) and overall survival (OS)., Results: EnhanceAR-Seq identified genomic alterations in the AR /enhancer locus in 45% of cases, including a 40% rate of AR enhancer amplification. Patients with AR /enhancer alterations had significantly worse PFS and OS than those without (6-month PFS: 30% vs. 71%, P=0.0002 ; 6-month OS: 59% vs. 100%, P=0.0015 ). AR /enhancer alterations in plasma cfDNA detected 18 of 23 resistant cases (78%) and outperformed the CTC AR-V7 assay which was also run on a subset of patients., Conclusion: cfDNA-based AR locus alterations, including of the enhancer, are strongly associated with resistance to AR-directed therapy and significantly worse survival. cfDNA analysis using EnhanceAR-Seq may enable more precise risk stratification and personalized therapeutic approaches for metastatic prostate cancer.

Circulating tumor DNA (ctDNA) is a component of cell-free DNA that is shed by malignant tumors into the bloodstream and other bodily fluids. Levels of ctDNA are typically low, particularly in patients with localized disease, requiring highly sophisticated methods for detection and quantification. Multiple liquid biopsy methods have been developed for ctDNA analysis in solid tumor malignancies and are now enabling detection and assessment of earlier stages of disease, post-treatment molecular residual disease (MRD), resistance to targeted systemic therapy, and tumor mutational burden. Understanding ctDNA biology, mechanisms of release, and clearance and size characteristics, in conjunction with the application of molecular barcoding and targeted error correction, have increased the sensitivity and specificity of ctDNA detection techniques. Combinatorial approaches including integration of ctDNA data with circulating protein biomarkers may further improve assay sensitivity and broaden the scope of ctDNA applications. Circulating viral DNA may be utilized to monitor disease in some virally induced malignancies. In spite of increasingly accurate methods of ctDNA detection, results need to be interpreted with caution given that somatic mosaicisms such as clonal hematopoiesis of indeterminate potential (CHIP) may give rise to genetic variants in the bloodstream unrelated to solid tumors, and the limited concordance observed between different commercial platforms. Overall, highly precise ctDNA detection and quantification methods have the potential to transform clinical practice via non-invasive monitoring of solid tumor malignancies, residual disease detection at earlier timepoints than standard clinical and/or imaging surveillance, and treatment personalization based on real-time assessment of the tumor genomic landscape.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

The p68 DEAD box helicases comprise a widely conserved protein family involved in a large range of biological processes including transcription, splicing and translation. The genome of the ciliate Tetrahymena thermophile encodes two p68-like helicases, Drh1p and Lia2p. We show that DRH1 is essential for growth and completion of development. In growing cells, Drh1p is excluded from the nucleus and accumulates near cortical basal bodies. In contrast, during sexual reproduction, this protein localizes to meiotic micronuclei, initially in punctate foci in regions where centromeres and telomeres are known to reside and later in post-zygotic differentiating somatic macronuclei. Differentiation of the macronuclear genome involves extensive DNA rearrangements including fragmentation of the five pairs of germline-derived chromosomes into 180 chromosomal sub-fragments that are stabilized by de novo telomere deletion. In addition, thousands of internal eliminated sequences (IESs) are excised from loci dispersed throughout the genome. Strains with DRH1 deleted from the germline nuclei, which do not express the protein during post-zygotic development, fail to fragment the developing macronuclear chromosomes. IES excision still occurs in the absence of DRH1 zygotic expression; thus, Drh1p is the first protein found to be specifically required for chromosome breakage but not DNA elimination., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON.

The malaria merozoite invades erythrocytes in the vertebrate host. Iterative rounds of asexual intraerythrocytic replication result in disease. Proteases play pivotal roles in erythrocyte invasion, but little is understood about their mode of action. The Plasmodium falciparum malaria merozoite surface sheddase, PfSUB2, is one such poorly characterized example. We have examined the molecular determinants that underlie the mechanisms by which PfSUB2 is trafficked initially to invasion-associated apical organelles (micronemes) and then across the surface of the free merozoite. We show that authentic promoter activity is important for correct localization of PfSUB2, likely requiring canonical features within the intergenic region 5' of the pfsub2 locus. We further demonstrate that trafficking of PfSUB2 beyond an early compartment in the secretory pathway requires autocatalytic protease activity. Finally, we show that the PfSUB2 transmembrane domain is required for microneme targeting, while the cytoplasmic domain is essential for surface translocation of the protease to the parasite posterior following discharge from micronemes. The interplay of pre- and post-translational regulatory elements that coordinate subcellular trafficking of PfSUB2 provides the parasite with exquisite control over enzyme-substrate interactions., (© 2013 The Authors. Traffic published by John Wiley & Sons Ltd.)

Background: Adenotonsillectomy (AT) is indicated for children with obstructive sleep disordered breathing; however it has associated well-documented morbidity. A subtotal reduction AT has made a resurgence overseas, given a significantly reduced morbidity. This study hypothesized that full AT would provide a greater improvement in quality of life (QOL) when compared with a subtotal reduction AT (SRAT) in children with obstructive sleep disordered breathing., Methods: This cohort study used a single surgeon consecutive series of 181 children from the database of the senior author (ASC) following full AT (n= 118) or SRAT (n= 63). QOL was measured by the Glasgow Children's Benefit Inventory (GCBI), which was mailed to parents 3 months to 2 years post-operatively., Results: Ninety-one of the 155 (59%) questionnaires were returned. There was an increase in QOL for children following AT (GCBI Total =+41.5) and SRAT (GCBI Total =+49.5). A significant increase in QOL was noted for all four domains of the GCBI. The GCBI total and four domains had no statistically significant difference in the improvement of scores by the two surgical groups., Conclusion: In this study, an SRAT provides identical post-operative QOL outcomes to full AT when performed for sleep disordered breathing in children. This adds to the evidence that in the absence of infective episodes, SRAT can be considered as a lower risk alternative to full AT., (© 2011 The Authors. ANZ Journal of Surgery © 2011 Royal Australasian College of Surgeons.)

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full-length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion., (© 2010 Blackwell Publishing Ltd.)

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl glutamate pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized through modifications at the 4-position of the pyridine ring and the terminal nitrogen of the piperazine ring, leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 47s with good human PRP potency, selectivity, in vivo efficacy, and oral bioavailability. Compound 47s was selected for further preclinical evaluations.

Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the 4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over candidate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency and improved pharmacokinetic profiles in both the rat and dog., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Objectives: To compare pepsin, carbonic anhydrase III (CAIII), cyclooxygenase-2 (COX-2) and mucin 5AC (MUC5AC) expression in children with adenoid hypertrophy and normal controls., Design: A non-randomised, controlled prospective study., Setting: Two paediatric hospitals in Adelaide, South Australia., Participants: Children aged 2-10 years, 21 undergoing adenoidectomy and 12 controls undergoing routine dental surgery., Main Outcome Measures: We measured expression of pepsin, CAIII, COX-2 and MUC5AC levels by real-time RT-PCR, immunohistochemistry, and Western blot to determine any difference between children with hyperplastic adenoids and controls., Results: Pepsin was not detected in any study or control adenoid by immunohistochemistry or Western blot. Real-time RT-PCR analysis showed a statistically significant difference between groups with respect to COX-2 (P = 0.027) and MUC5AC (P = 0.02) but no difference in CAIII expression (P = 0.414). A significant correlation was also found between COX-2 and MUC5AC expression (Kendall Tau = 0.4, P = 0.005)., Conclusion: Our results suggest that the biochemical changes seen in adenoid hypertrophy are different to those seen in reflux-affected tissues. The decreased COX-2 and MUC5AC expression may be due to squamous metaplasia and other inflammatory changes associated with adenoid hypertrophy. Our findings infer there is little evidence of reflux being a major contributory factor in the pathophysiology of adenoidal hypertrophy.

Objective: To assess current tonsillectomy practice among Australian otolaryngologists., Study Design: An audit based on an anonymous 19-item postal questionnaire on tonsillectomy technique and perioperative management sent to all Australian otolaryngology specialists., Subjects and Methods: Two hundred eighty-four otolaryngologists registered with the Australian Society of Otolaryngology-Head and Neck Surgery database were sent the questionnaire., Results: A 72.5 percent response rate was obtained. Monopolar diathermy was the most common technique for dissection (45%) and hemostasis (54%). Bipolar diathermy was used for hemostasis in 20 percent. Cold-steel dissection was routinely used by 36 percent, ties were used for hemostasis only by 11 percent of surgeons. The use of local anesthetic, dexamethasone, and postoperative antibiotics was 45 percent, 40 percent, and 20 percent, respectively. Seventy-six percent of surgeons always observed tonsil patients overnight., Conclusion: Australian surgeons still use monopolar diathermy as their preferred technique for tonsillectomy. Local anesthetic, dexamethasone, and postoperative antibiotics are used infrequently, and fewer than 1:4 surgeons perform day-case tonsillectomy.

Objective: To establish if there is a learning curve for coblation tonsillectomy., Study Design: Regression analysis of data obtained from surgeons identified from the Australian Tonsillectomy Survey., Subjects and Methods: Thirty otolaryngologists were invited to contribute audit data. Data were stratified into groups of 10 procedures and analysed with regression analysis., Results: Nineteen (70%) surgeons responded. Complete data were obtained for 1700 cases and return to theatre data on 2062 cases. There was a significant learning curve with respect to both primary (P = 0.050) and secondary (P = 0.028) hemorrhage rates. Mean rates were 0.3% (95% CI 0.1% to 0.7%) and 2.1% (95% CI 1.5% to 2.9%) for primary and secondary bleeds, respectively, with return to theatre in 0.2% (95% CI 0.1% to 0.5%) and 1.3% (95% CI 0.9% to 1.9%), respectively., Conclusion: The introduction of coblation tonsillectomy into Australia was associated with a statistically significant learning curve with respect to both primary and secondary hemorrhage rates.

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface "sheddase," but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite.

Purpose: Otitis media is the most common condition diagnosed by pediatricians and is estimated to affect approximately 70% of the pediatric population. The goal of this study was to evaluate the effectiveness of otoscopy and multifrequency tympanometry (MFT) for diagnosis of otitis media in children., Method: Twenty-one children, age 1 to 10 years, who were seeking medical treatment for suspected middle ear disease were selected to participate. Data were collected prior to myringotomy to determine the sensitivity and specificity rates of the following otologic and audiologic measures: (a) pneumatic otoscopy, (b) conventional tympanometry, and (c) MFT. For this study, the "gold standard," myringotomy, was used along with pneumatic otoscopy to determine the effectiveness, sensitivity, and specificity of conventional 226-Hz tympanometry, 678-Hz tympanometry, and 1000-Hz tympanometry to predict middle ear disease., Results: The diagnoses provided with pneumatic otoscopy and tympanometry were both similar, agreeing in diagnosis 80%-100% of the time. The diagnoses from 678-Hz and 1000-Hz tympanometry were nearly equal and proved to detect abnormality at a higher rate., Conclusions: MFT is recommended on a routine basis with children having a history of otitis media, or else abnormal or notched 226-Hz tympanograms. Further research with a larger sample size will illuminate the possible predictive potential of MFT in otitis media.

Differential mRNA display RT-PCR (DD RT-PCR) offers a tool to identify genes which are regulated or responsive to certain receptors or chemicals such as dioxin (TCDD). Treatment of Hep G2 cells with TCDD followed by DD analysis of a gel with series of different primers revealed a significantly different pattern from the control for a number of mRNAs. The differentially displayed mRNAs were isolated and reamplified. A GenBank search of four mRNAs revealed two known and two unknown sequences. Northern blot analysis revealed that two known sequences, fibrinogen gamma chain and plastin mRNAs were down regulated by TCDD in a time-dependent manner, whereas two unknown mRNAs were induced by TCDD treatment. The function of these genes in TCDD toxicity is not known; however, the application of DD RT-PCR in the studies of TCDD-induced responses could be very useful in the discovery of other unknown genes important for TCDD toxicity.

The thiazolidinediones are a class of antidiabetic compounds that increase the sensitivity of target tissues to insulin. An earlier study has shown that these compounds enhance the insulin-stimulated differentiation of 3T3-L1 cells and up-regulate expression of differentiation-dependent genes. We have observed that the mRNA encoding the adipocyte fatty acid-binding protein (aFABP) increases shortly after incubation of cells with pioglitazone, a thiazolidinedone analogue. The drug was found to enhance, in a time- and dose-dependent fashion, the expression of a chimeric gene that was constructed by fusing the aFABP promoter upstream of the chloramphenicol acetyltransferase (CAT) gene. To localize the sequence within the promoter that is responsive to pioglitazone, a series of chimeric genes containing sections of the aFABP promoter fused to the CAT gene were analyzed after transfection of 3T3-L1 cells. A section of DNA located at -5.2 kilobases and known to encompass a tissue-specific and differentiation-dependent enhancer element was found to confer responsiveness to the drug. Analysis of sequences in this region of the aFABP promoter by DNA gel retardation assays revealed the presence of a protein in nuclear extracts from drug-treated cells that bound to a specific sequence (ARE-6). The presence of the protein could be demonstrated in differentiated adipocytes, but the protein was present at only low levels in preadipocytes. Treatment of preadipocytes with pioglitazone resulted in the precocious appearance of this protein in nuclear extracts. Multiple copies of the ARE-6 sequence inserted upstream of a heterologous promoter linked to the CAT gene conferred pioglitazone responsiveness. The experiments reported in this study establish that the insulin-sensitizing agent pioglitazone up-regulates expression of the aFABP gene through an element located within a region of DNA responsible for tissue-specific and differentiation-dependent expression of the gene.

The enzyme, glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49), has long been considered and studied as the archetypical X-linked "housekeeping" enzyme that is present in all cells, where it plays the key role in regulating carbon flow through the pentose phosphate pathway. Specifically, the enzyme catalyzes the first reaction in the pathway leading to the production of pentose phosphates and reducing power in the form of NADPH for reductive biosynthesis and maintenance of the redox state of the cell. It was in this latter function that the crucial importance of the enzyme was first appreciated with the description of the human deficiency syndrome. While the gene can be considered to be a constitutively expressed "housekeeping" gene in many tissues, there are several other tissues (liver, adipose, lung, and proliferating cells) wherein modulation of cellular G6PDH activity represents an important component of the integrated response to external stimuli (hormones, growth factors, nutrients, and oxidant stress). In this regard, adaptive regulation of G6PDH has been found to be exerted at transcriptional and posttranscriptional levels. However, the regulation observed is tissue-specific, which elicits the central question of this review, "How can the G6PDH gene be constitutively expressed in some tissues while displaying adaptive regulation in others when there exists a single transcription unit for the gene?" Future studies utilizing cloned genomic fragments of the human and other mammalian G6PDH genes should provide answers to this question.

A 935 bp fragment of the rat glucose-6-phosphate dehydrogenase (G6PDH) gene containing promoter activity was isolated using the polymerase chain reaction (PCR). This fragment was sequenced and primer extension analysis showed a transcription initiation site in agreement with the human and mouse genes. Computer analysis of the sequence showed a 60% and 78% similarity to the human and mouse G6PDH sequences, respectively. A TATA box element, TTAAAT, was found and shown to be 100% similar to the human and mouse TATA box elements. Based on sequence comparison, some putative transcriptional regulatory elements were also found.

Pioglitazone, a thiazolidinedione, is a novel antidiabetic compound that can lower blood glucose in diabetic rodents by increasing insulin sensitivity in target tissues. We have previously demonstrated that pioglitazone can enhance the insulin- or insulin-like growth factor-1-regulated differentiation of 3T3-L1 cells, a cell line that undergoes morphological and biochemical differentiation to mature adipocytes [Mol. Pharmacol. 41:393-398 (1992)]. In this study, we have examined the effect of pioglitazone on the expression of the adipocyte fatty acid-binding protein (aFABP) in ob/ob mice and 3T3-L1 cells. Administration of the drug to mice was observed to cause a dose-dependent increase in aFABP mRNA expression in epididymal fat, which was correlated with a decrease in blood glucose and insulin levels. Treatment of 3T3-L1 cells with pioglitazone enhanced aFABP expression in a time-dependent fashion. To explore a possible direct effect of pioglitazone on aFABP expression, a chimeric gene was constructed containing the aFABP promoter fused upstream of the bacterial reporter gene for chloramphenicol acetyltransferase. After transfection into 3T3-L1 cells and selection of stable transformants, regulation of the chimeric gene was studied. Pioglitazone, in combination with insulin or insulin-like growth factor-1, was observed to elicit a dose-dependent increase in expression, indicating a role for pioglitazone in regulating transcription of the aFABP gene. Several thiazolidinedione analogs were tested for their ability to induce the expression of the chimeric gene, and it was found that activity in this assay paralleled the structure-activity relationships observed for enhancement of 3T3-L1 cell differentiation. These observations on control of aFABP gene expression by pioglitazone suggest possible mechanisms by which cellular sensitivity to insulin may be regulated.

Our previous studies have demonstrated that platelet-derived growth factor (PDGF) modulated cellular responses to interleukin-1 (IL-1). In this communication, we show that PDGF regulates expression of IL-1 receptor (IL-1 R) gene. Treatment of quiescent cultures of Balb/c 3T3 fibroblasts with PDGF produced 20-30-fold stimulation of IL-1 R mRNA with a concomitant increase in cell surface 125I-binding. IL-1 R mRNA accumulation occurred after an initial lag period and with a time course preceding the increase in 125I-IL-1 binding to cells. Induction of IL-1 R mRNA was blocked by inhibitors of protein synthesis, suggesting that a product of a gene expressed immediately after PDGF addition is required for IL-1 R gene expression. These latter data provide evidence for an ordered sequence of expression of PDGF-inducible "immediate early" gene(s) and IL-1 R gene. These results suggest that in connective tissues, PDGF may be an important determinant in initiating and maintaining cellular responses to IL-1. Such responses may have important consequences in the actions of IL-1 under normal and pathological conditions such as arthritis and atherosclerosis.

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.

The levels of four ribosomal protein (rp) mRNAs in different mutant strains were determined by hydridization of radiolabeled cloned genes to RNA fractionated on CH3HgOH gels and transferred to DBM paper. Two ribosomal protein genes (rp 51 and rp 52) controlled by the locus RNA2 have dramatically decreased mRNA levels after a shift-up to the nonpermissive temperature in a strain carrying the rna2 mutation (ts368). Two ribosomal protein genes not controlled by the RNA2 locus and several control nonribosomal protein genes are relatively unaffected by the temperature shift in this strain. Other genes in the vicinity of one of the rna2-sensitive ribosomal protein genes (th rp 51 gene) are insensitive to the rna2 gene product, suggesting that all ribosomal protein genes do not occur in clusters and that the RNA2 gene product does not affect a large region of chromatin. In ts368 at the nonpermissive temperature, the concentration of higher molecular weight transcripts complementary to the rp 51 and the rp 52 plasmids is increased. Analysis of the rp 51 plasmid transcripts reveals that the temperature-induced higher molecular weight transcripts differ from the mature rp 51 mRNA by the presence of an intron. This observation and the kinetics with which the concentration of the various rp 51 transcripts change after a temperature shift suggest that the effect of rna2 may be at the level of processing of rp mRNA.