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Urinary tract infections (UTIs) are common, recurrent infections that can be mild to life-threatening. The continued emergence of antibiotic resistance, together with our increasing understanding of the detrimental effects conferred by broad-spectrum antibiotic use on the health of the beneficial microbiota of the host, has underscored the weaknesses in our current treatment paradigm for UTIs. In this Review, we discuss how recent microbiological, structural, genetic and immunological studies have expanded our understanding of host-pathogen interactions during urinary tract pathogenesis. These basic scientific findings have the potential to shift the strategy for UTI treatment away from broad-spectrum antibiotics targeting conserved aspects of bacterial replication towards pathogen-specific antibiotic-sparing therapeutics that target core determinants of bacterial virulence at the host-pathogen interface.

BACKGROUND Molecular genetic testing has raised a variety of policy issues, ranging from privacy to reimbursement. Recently, payment policies have become of paramount importance as Medicare implemented the first significant change to test pricing since 1984 and announced a broad national coverage policy for the use of next-generation sequencing (NGS) in cancer patients that contains significant restrictions. Regulatory and oversight concerns have been important topics for discussion as the US Food and Drug Administration (FDA), Congress, and stakeholders have focused on new approaches to regulation of laboratory-developed tests (LDTs). Patents on gene sequences and relationships between genetic variants and clinical phenotypes have been points of contention since the field's inception. Two Supreme Court cases invalidated patents on gene sequences and biological relationships, ushering in the era of NGS and precision medicine. However, a recent legislative proposal threatens to reverse these gains and restore gene patents as barriers to progress in genetic and genomic testing and the implementation of genomic medicine. CONTENT This review discusses current issues in payment policy, laboratory oversight, and gene patenting and their potential impacts on genetic and genomic testing. SUMMARY Coverage and reimbursement policies present serious challenges to genetic and genomic testing. The potential for FDA regulation of LDTs looms as a significant threat to diagnostic innovation, patient access, and the viability of molecular genetic testing laboratories. Changes in patent law could cause gene patents to reemerge as barriers to the advancement of genomic medicine.

The F9/Yde/Fml pilus, tipped with the FmlH adhesin, has been shown to provide uropathogenic Escherichia coli (UPEC) a fitness advantage in urinary tract infections (UTIs). Here, we used X-ray structure guided design to optimize our previously described ortho-biphenyl Gal and GalNAc FmlH antagonists such as compound 1 by replacing the carboxylate with a sulfonamide as in 50. Other groups which can accept H-bonds were also tolerated. We pursued further modifications to the biphenyl aglycone resulting in significantly improved activity. Two of the most potent compounds, 86 (IC(50) = 0.051 μM) and 90 (IC(50) = 0.034 μM), exhibited excellent metabolic stability in mouse plasma and liver microsomes but showed only limited oral bioavailability (

The emergence of drug-resistant pathogens has led to a decline in the efficacy of traditional antimicrobial therapy. The rise in resistance has been driven by widespread use, and in some cases misuse, of antibacterial agents in treating a variety of infections. A growing body of research has begun to elucidate the harmful effects of broad-spectrum antibiotic therapy on the beneficial host microbiota. To combat these threats, increasing effort is being directed toward the development of precision antimicrobial therapeutics that target key virulence determinants of specific pathogens while leaving the remainder of the host microbiota undisturbed. This includes the recent development of small molecules termed “mannosides” that specifically target uropathogenic E. coli (UPEC). Mannosides are glycomimetics of the natural mannosylated host receptor for type 1 pili, extracellular appendages that promotes UPEC colonization in the intestine. Type 1 pili are also critical for colonization and infection in the bladder. In both cases, mannosides act as molecular decoys which potently prevent bacteria from binding to host tissues. In mice, oral treatment with mannosides simultaneously clears active bladder infection and removes intestinal UPEC while leaving the gut microbiota structure relatively unchanged. Similar treatment strategies successfully target other pathogens, like adherent-invasive E. coli (AIEC), an organism associated with Crohn’s disease (CD), in mouse models. While not without its challenges, antibiotic-sparing therapeutic approaches hold great promise in a variety of disease systems, including UTI, CD, otitis media (OM), and others. In this perspective we highlight the benefits, progress, and roadblocks to the development of precision antimicrobial therapeutics.

BACKGROUND The detection of mutated epidermal growth factor receptor (EGFR) in non–small cell lung cancer (NSCLC) with residual cell pellets derived from liquid-based cytology (LBC) samples (eg, endoscopic ultrasound–guided fine-needle aspiration) has been validated with allele-specific polymerase chain reaction. The aim of this study was to validate next-generation sequencing (NGS) technology for detecting gene mutations with residual cell pellets from LBC. METHODS Archived DNA extracted from LBC samples of adenocarcinoma stored in PreservCyt with a known EGFR mutation status was retrieved. Genomic DNA was multiplex-amplified and enriched with Ion AmpliSeq Cancer Hotspot Panel v2 chemistry and the OneTouch 2 instrument; this was followed by semiconductor sequencing on the Ion Personal Genome Machine platform. The mutation hotspots of 6 NSCLC-related genes (BRAF, EGFR, ERBB2, KRAS, MET, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α [PIK3CA]) were analyzed with NextGENe and Torrent Suite bioinformatics tools. RESULTS The commonly identified EGFR sequence changes, including 4 L858R mutations, 3 exon 19 deletions, and 1 exon 20 insertion, were in 100% concordance between the assay platforms. Less common NSCLC variants were also found in the mutation hotspots of ERBB2, KRAS, MET, and PIK3CA genes. CONCLUSIONS NSCLC mutation analysis using NGS can be successfully performed on residual cell pellets derived from LBC samples. This approach allows the simultaneous examination of multiple mutation hotspots in a timely manner to improve patient care. Cancer Cytopathol 2017;125:178–187. © 2016 American Cancer Society.

Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the O-glycosidic bond (O-mannosides). Herein, we disclose that compounds having the O-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked C-mannosides that show improved pharmacokinetic (PK) properties relative to O-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the R-hydroxy isomer has a 60-fold increase in potency. This new class of C-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI.

Clinical utility describes the benefits of each laboratory test for that patient. Many stakeholders have adopted narrow definitions for the clinical utility of molecular testing as applied to targeted pharmacotherapy in oncology, regardless of the population tested or the purpose of the testing. This definition does not address all of the important applications of molecular diagnostic testing. Definitions consistent with a patient-centered approach emphasize and recognize that a clinical test result's utility depends on the context in which it is used and are particularly relevant to molecular diagnostic testing because of the nature of the information they provide. Debates surrounding levels and types of evidence needed to properly evaluate the clinical value of molecular diagnostics are increasingly important because the growing body of knowledge, stemming from the increase of genomic medicine, provides many new opportunities for molecular testing to improve health care. We address the challenges in defining the clinical utility of molecular diagnostics for inherited diseases or cancer and provide assessment recommendations. Starting with a modified analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications model for addressing clinical utility of molecular diagnostics with a variety of testing purposes, we recommend promotion of patient-centered definitions of clinical utility that appropriately recognize the valuable contribution of molecular diagnostic testing to improve patient care.

Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.

Gene patents have been highly controversial in clinical diagnostics. Proponents claim that these patents promote gene discovery and encourage the production of novel diagnostic tests. Opponents argue that patents are unnecessary for discovery, and that they raise costs, decrease patient access, and harm innovation in the field of molecular pathology. In two recent Supreme Court cases, Mayo v. Prometheus and Association for Molecular Pathology v. Myriad Genetics, the Court ruled that biological correlations and human DNA sequences represent natural laws that cannot be patented. These cases appear to have eliminated patent-based monopolization of testing for mutations in human genes and genotype–phenotype relationships, and will help facilitate the introduction of large-scale sequencing into clinical practice. The Supreme Court has thereby encouraged the advancement, development, and implementation of personalized medicine.

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal “tails” were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections., IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis.

The Center for Medical Technology Policy and the Molecular Evidence Development Consortium gathered a diverse group of more than 50 stakeholders to develop consensus on a core set of data elements and values essential to understanding the clinical utility of molecularly targeted therapies in oncology.

The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group found insufficient evidence to recommend prostate cancer antigen 3 (PCA3) testing to inform decisions for when to rebiopsy previously biopsy-negative patients for prostate cancer or to inform decisions to conduct initial biopsies for prostate cancer in at-risk men (e.g., previous elevated prostate-specific antigen test or suspicious digital rectal examination).The EGAPP Working Group found insufficient evidence to recommend PCA3 testing in men with cancer-positive biopsies to determine if the disease is indolent or aggressive in order to develop an optimal treatment plan.Based on the available evidence, the overall certainty of clinical validity to predict the diagnosis of prostate cancer using PCA3 is deemed "low." The EGAPP Working Group discourages clinical use for diagnosis unless further evidence supports improved clinical validity.Based on the available evidence, the overall certainty of net health benefit is deemed "low." The EGAPP Working Group discourages clinical use unless further evidence supports improved clinical outcomes.It has been suggested that PCA3 testing in the general male population might lead to earlier diagnosis and management changes (e.g., earlier detection and earlier initiation or higher rates of medical interventions) that improve outcomes. EGAPP Working Group found no direct evidence to support this possibility, so we sought indirect evidence aimed at documenting the extent to which PCA3 testing alters prostate cancer diagnosis or management, alone and in combination with traditional clinical management factors, and the extent to which this testing improves health outcomes.Assay-related evidence was deemed adequate for the PROGENSA PCA3 assay approved by the US Food and Drug Administration, available from Gen-Probe. Very few studies were available that investigated preanalytical effects, analytical performance, and diagnostic accuracy of other quantitative assays for PCA3.Evidence on clinical validity was rated inadequate to derive any conclusions about performance of PCA3 testing to inform decisions for when to rebiopsy previously biopsy-negative patients for prostate cancer, or to inform decisions to conduct initial biopsies for prostate cancer in at-risk men (e.g., previous elevated prostate-specific antigen test or suspicious digital rectal examination). Furthermore, there was little evidence to derive any conclusions about performance of PCA3 testing in men with cancer-positive biopsies to determine if the disease is indolent or aggressive in order to develop an optimal treatment plan.No studies were available to provide direct evidence on the balance of benefits and harms related to PCA3 testing for diagnosis and management in the general male population. Evidence for other populations (e.g., high risk) was not evaluated in the review.Early diagnosis of prostate cancer is central to minimizing morbidity and mortality. Prevention of prostate cancer mortality is a public health priority. Improvements in outcomes associated with PCA3 testing could have important impacts.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Since 2006, the US Food and Drug Administration, Congress, and other policymakers have explored the appropriate way to guarantee the clinical and analytical validity of laboratory-developed tests. In the past, the Association for Molecular Pathology has publicly urged the Food and Drug Administration to exercise caution in implementing regulatory changes that could potentially hinder innovation or interfere with the practice of medicine. In 2012, the Association for Molecular Pathology Professional Relations Committee chose to develop this paper with the goal of outlining the best methods for ensuring appropriate oversight and validation of molecular diagnostic procedures. At the conclusion of this process, the workgroup reaffirmed the Association's previous position that the Centers for Medicare and Medicaid Services Clinical Laboratory Improvement Amendments program can provide the appropriate level of oversight for the vast majority of diagnostic tests.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

To provide an update on recent revisions to Evaluation of Genomic Applications in Practice and Prevention (EGAPP) methods designed to improve efficiency, and an assessment of the implications of whole genome sequencing for evidence-based recommendation development. Improvements to the EGAPP approach include automated searches for horizon scanning, a quantitative ranking process for topic prioritization, and the development of a staged evidence review and evaluation process. The staged process entails (i) triaging tests with minimal evidence of clinical validity, (ii) using and updating existing reviews, (iii) evaluating clinical validity prior to analytic validity or clinical utility, (iv) using decision modeling to assess potential clinical utility when direct evidence is not available. EGAPP experience to date suggests the following approaches will be critical for the development of evidence based recommendations in the whole genome sequencing era: (i) use of triage approaches and frameworks to improve efficiency, (ii) development of evidence thresholds that consider the value of further research, (iii) incorporation of patient preferences, and (iv) engagement of diverse stakeholders. The rapid advances in genomics present a significant challenge to traditional evidence based medicine, but also an opportunity for innovative approaches to recommendation development.

The detection of mutated epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) with residual cell pellets derived from liquid-based cytology (LBC) samples (eg, endoscopic ultrasound-guided fine-needle aspiration) has been validated with allele-specific polymerase chain reaction. The aim of this study was to validate next-generation sequencing (NGS) technology for detecting gene mutations with residual cell pellets from LBC.Archived DNA extracted from LBC samples of adenocarcinoma stored in PreservCyt with a known EGFR mutation status was retrieved. Genomic DNA was multiplex-amplified and enriched with Ion AmpliSeq Cancer Hotspot Panel v2 chemistry and the OneTouch 2 instrument; this was followed by semiconductor sequencing on the Ion Personal Genome Machine platform. The mutation hotspots of 6 NSCLC-related genes (BRAF, EGFR, ERBB2, KRAS, MET, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α [PIK3CA]) were analyzed with NextGENe and Torrent Suite bioinformatics tools.The commonly identified EGFR sequence changes, including 4 L858R mutations, 3 exon 19 deletions, and 1 exon 20 insertion, were in 100% concordance between the assay platforms. Less common NSCLC variants were also found in the mutation hotspots of ERBB2, KRAS, MET, and PIK3CA genes.NSCLC mutation analysis using NGS can be successfully performed on residual cell pellets derived from LBC samples. This approach allows the simultaneous examination of multiple mutation hotspots in a timely manner to improve patient care. Cancer Cytopathol 2017;125:178-187. © 2016 American Cancer Society.

Bacterial adherence to host tissue is an essential process in pathogenesis, necessary for invasion and colonization and often required for the efficient delivery of toxins and other bacterial effectors. As existing treatment options for common bacterial infections dwindle, we find ourselves rapidly approaching a tipping point in our confrontation with antibiotic-resistant strains and in desperate need of new treatment options. Bacterial strains defective in adherence are typically avirulent and unable to cause infection in animal models. The importance of this initial binding event in the pathogenic cascade highlights its potential as a novel therapeutic target. This article seeks to highlight a variety of strategies being employed to treat and prevent infection by targeting the mechanisms of bacterial adhesion. Advancements in this area include the development of novel antivirulence therapies using small molecules, vaccines, and peptides to target a variety of bacterial infections. These therapies target bacterial adhesion through a number of mechanisms, including inhibition of pathogen receptor biogenesis, competition-based strategies with receptor and adhesin analogs, and the inhibition of binding through neutralizing antibodies. While this article is not an exhaustive description of every advancement in the field, we hope it will highlight several promising examples of the therapeutic potential of antiadhesive strategies.

Adhesion to biotic and abiotic surfaces is an essential step in the infectious cycle of many human pathogens. To this end, both Gram-positive and Gram-negative bacteria have developed a variety of strategies to overcome innate clearance mechanisms and persist within the host. The expression and biogenesis of Chaperone-Usher Pathway (CUP) pili represent one such strategy. In addition to mediating the formation of bacterial biofilms, these proteinaceous extracellular appendages contain, at their tip, a two-domain adhesin that is composed of an N-terminal lectin domain and a C-terminal pilin domain.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.

In 2008, the Association for Molecular Pathology (AMP)2 Economic Affairs Committee (EAC), under the leadership of the late Dr. Jeffrey Kant, embarked on a project to design new Current Procedural Terminology (CPT) codes for identification and billing of molecular pathology services. At that time, payers were confronted with a dizzying array of highly technical method-based CPT codes that described the various steps used in performing molecular assays. Examples included “83890 isolation or extraction, each nucleic acid type (i.e., DNA or RNA)”; “83898 amplification, target, each nucleic acid sequence”; “83904 mutation identification by sequencing, single segment, each segment”; and “83912 interpretation and report.” Laboratories “stacked” these codes on claim forms along with numerals that specified the number of times each step was performed. Payers could only guess at what the particular test was or why it was performed. Use of the CPT stacking codes was not uniform, and there was no way to ensure that tests were properly coded. Moreover, if more than one test was performed on a sample, Medicare Administrative Contractors (MACs) were unable to allocate CPT codes between tests. Finally, this coding structure left open the possibility that reimbursement rates for individual steps could influence assay design. Despite their shortcomings, the stacking codes had a powerful redeeming feature. Molecular pathology was characterized by both rapid growth and continual technological change. The stacking code format offered great flexibility and an ability to readily accommodate new tests and assay designs, benefitting patients and helping to advance the field. The AMP EAC's efforts culminated in production of a whitepaper that proposed an innovative approach to coding molecular pathology procedures in genetics, oncology, and histocompatibility. This document was submitted to the American Medical Association (AMA) CPT Editorial Panel for consideration (1). In response, the AMA convened a workgroup “to propose solutions …

The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 μM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.

Roger D Klein, MD, JD, is the Medical Director of Molecular Oncology at BloodCenter of Wisconsin (WI, USA). He previously served as the Medical Director of Molecular Diagnostics at H Lee Moffitt Cancer Center (FL, USA), and Assistant Professor in the Department of Oncologic Sciences at University of South Florida Medical School (FL, USA). He earned his undergraduate and medical degrees and completed an internship in Internal Medicine at Case Western Reserve University (OH, USA). He completed residency training in Laboratory Medicine, together with fellowships in Medical Microbiology and Molecular Genetics at Yale University School of Medicine (CT, USA) followed by a fellowship in Molecular Genetic Pathology at Mayo Clinic (MN, USA). In addition, Dr Klein earned a law degree from Yale Law School, where he was named an Olin Fellow for Law and Public Policy Studies and served as an articles editor of the Yale Journal on Regulation. He is board-certified in Clinical Pathology and Molecular Genetic Pathology. Dr Klein is a member of the CDC’s Evaluation of Genomic Applications in Practice and Prevention Working Group (EGAPP), and is a consultant to the US FDA’s Clinical and Molecular Genetics Advisory Panel. He has served on the College of American Pathologists ‘Biochemical and Molecular Genetics’ and ‘Point-of-Care Testing’ Resource Committees, and is currently a member of the College of American Pathologists Molecular Oncology Resource Committee. He also serves on the Association for Molecular Pathology’s current procedural terminology (CPT) Coding and Professional Relations Committees. Dr Klein’s academic and clinical efforts focus on the translation of molecular genetic knowledge into clinical diagnostic tests for the evaluation and management of hematopoietic and solid tumor malignancies, and hereditary cancer syndromes. This work includes the assessment of the clinical validity, clinical utility and the optimal and appropriate use of molecular genetic tests. In addition, Dr Klein has an active research program involving the ethical, legal and social implications of the Human Genome Project, with particular emphases on the areas of intellectual property and the regulation of in vitro diagnostics and clinical laboratories. He has multiple publications in these areas.

Germline mutations in the genes encoding the B (SDHB) and D (SDHD) subunits of the heterotetrameric protein succinate dehydrogenase (mitochondrial complex II) are important causes of inherited and apparently sporadic paragangliomas. In an effort to further investigate the role of these genes in malignant sympathetic paragangliomas and adrenal pheochromocytomas, we screened a series of tumors for mutations in SDHB and SDHD. Mutation testing was performed on DNA extracted from formalin-fixed, paraffin-embedded tumors and associated normal tissues by polymerase chain reaction amplification and direct sequencing of the coding regions and intron-exon junctions of the SDHB and SDHD genes. Among 16 malignant paragangliomas with proven metastases, 6 (38%) had mutations in SDHB (2 nonsense, 1 splice site, 1 insertion causing a frameshift, and 2 presumably deleterious missense mutations). Probable deleterious SDHB variants were also detected in 5 (45%) of 11 paragangliomas without known metastatic disease (1 splice site, 1 deletion causing a frameshift, and 3 missense changes). In 12 malignant pheochromocytomas, 1 SDHD and no SDHB mutations were identified. The identical SDHB mutation was detected in DNA extracted from accompanying normal tissue for each of the 10 cases on which this analysis was performed. An excess of SDHB mutations in paragangliomas versus pheochromocytomas was found, with no difference in the frequency of mutations in malignant versus benign paragangliomas. The disparate mutational spectra in malignant paragangliomas and pheochromocytomas may reflect differences in underlying tumor biology.

Uropathogenic E. coli (UPEC) employ the mannose-binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half-life in vivo. In a rational design strategy, we obtained an X-ray structure of lead mannosides and then designed mannosides with improved drug-like properties. We show that cyclizing the carboxamide onto the biphenyl B-ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2-methyl-4-(1-oxo-1,2-dihydroisoquinolin-7-yl)phenyl α-d-mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N-Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22, as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria-mediated hemagglutination and prevent biofilm formation by UPEC with single-digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date.

Clinical utility describes the benefits of each laboratory test for that patient. Many stakeholders have adopted narrow definitions for the clinical utility of molecular testing as applied to targeted pharmacotherapy in oncology, regardless of the population tested or the purpose of the testing. This definition does not address all of the important applications of molecular diagnostic testing. Definitions consistent with a patient-centered approach emphasize and recognize that a clinical test result's utility depends on the context in which it is used and are particularly relevant to molecular diagnostic testing because of the nature of the information they provide. Debates surrounding levels and types of evidence needed to properly evaluate the clinical value of molecular diagnostics are increasingly important because the growing body of knowledge, stemming from the increase of genomic medicine, provides many new opportunities for molecular testing to improve health care. We address the challenges in defining the clinical utility of molecular diagnostics for inherited diseases or cancer and provide assessment recommendations. Starting with a modified analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications model for addressing clinical utility of molecular diagnostics with a variety of testing purposes, we recommend promotion of patient-centered definitions of clinical utility that appropriately recognize the valuable contribution of molecular diagnostic testing to improve patient care.

Eighty-one percent of U.S. laboratories operate under a certificate of waiver or a provider-performed microscopy certificate, which allow them to perform a strictly defined subset of tests essentially without oversight. Waived tests, under the Clinical Laboratory Improvement Amendments statute, are required to be “simple” and to have an “insignificant risk of an erroneous result.” The number of analytes and individual test systems available as waived methods is large and increasing. Infectious disease tests available in waived format include Helicobacter pylori antibody, HIV-1 and 2 antibody, Influenza A/B antigen, Lyme antibody screen, Monospot, respiratory syncytial virus (RSV) antigen, RSV antibody, and Streptococcus group A antigen. Waived tests represent a compromise between access and quality of care. Quality problems in waived testing settings, demonstrated by state and Centers for Medicare and Medical Services (CMS) surveys, suggest that laboratory professionals could improve care by supporting waived testing laboratories, if systems to facilitate such interactions can be developed.

Purpose: This study determines which clinical features predict positive test results among samples submitted for DNA-based diagnostic nevoid basal cell carcinoma syndrome (NBCCS) testing, and further defines the mutational spectrum of the PTCH gene. Methods: DNA was extracted from peripheral blood leukocytes, and polymerase chain reaction products from exons 1 to 23 of the PTCH gene were directly sequenced. Pedigree phenotypic information was obtained by written questionnaire. Results: Among 106 presumably unrelated pedigrees, 44 independent mutations were found in 47 families. There were 11 nonsense mutations; 1 in-frame deletion; 17 deletions, 6 insertions, and 1 deletion-insertion that generated frameshifts; 5 splice-site mutations; 1 in-frame duplication; and 2 presumptive missense mutations. Twenty-seven of 46 pedigrees (58.7%) with two or more typical radiographic or pathologic features of NBCCS tested positive for PTCH mutations. Of these, 26 had jaw cysts in combination with other characteristics or neoplasms including basal cell carcinomas, palmar pits, skeletal abnormalities, ocular abnormalities, medulloblastomas, cardiac or ovarian fibromas, calcification of the falx cerebri, polydactyly, cleft lip and/or palate, and agenesis of the corpus callosum or other central nervous system malformations. None of the 13 pedigrees solely affected by multiple or early-onset basal cell carcinomas and none of the four pedigrees with jaw cysts alone had PTCH mutations. Conclusions: Pedigrees with multiple features of NBCCS were most likely to test positive for PTCH mutations. Pedigrees with multiple or early-onset basal cell carcinomas without other features of the disease did not test positive for PTCH mutations.

Purpose: Based on results of diagnostic MEN1 testing, we have attempted to further define the mutational spectrum of the MEN1 gene and the clinical features most frequently associated with MEN1 mutations. Methods: Mutation testing was performed on blood samples by PCR amplification and sequencing of exons 2 to 10 of the MEN1 gene and the corresponding intron-exon junctions. Pedigree phenotypic information was obtained by written questionnaire. Results: Among 288 presumably unrelated pedigrees, 73 independent mutations were found in 89 families. Five mutations were found in 2 pedigrees, and 4 mutations were seen in more than 2 pedigrees. There were 17 nonsense mutations (23.3%), 2 in-frame deletions (2.7%), 18 frameshift-deletion mutations (24.7%), 10 frameshift-insertion or -duplication mutations (13.7%), 13 splice-site mutations (17.8%), and 13 presumptive missense mutations (17.8%). Thirty-nine of 56 pedigrees with parathyroid and pancreatic islet neoplasia tested positive, compared with 4/24 and 8/32 pedigrees affected with hyperparathyroidism or hyperparathyroidism and pituitary tumors. MEN1 mutations were found in 6/20 sporadic patients, all of whom had both parathyroid and pancreatic neoplasms. Of 14 mutation-negative sporadic patients, 10 exhibited hyperparathyroidism and pituitary tumors without islet cell neoplasia. Somatic mosaicism was detected in 1 sporadic patient. Conclusion: Patients from pedigrees with hyperparathyroidism and pancreatic islet tumors are most likely to test positive for MEN1 mutations. Mutations are less often detected in patients from pedigrees with hyperparathyroidism alone or in combination with pituitary tumors without pancreatic islet neoplasia. Sporadic cases are less likely to test positive than familial cases, in part due to somatic mosaicism.

This article examines how local television news has been shown to misrepresent minorities. Minorities are portrayed more frequently as criminals than are Whites, and minority misrepresentation has been shown to be out of proportion to crime statistics. Several possible explanations are given for this phenomenon. Evidence exists that audiences are aware of misrepresentation and that news directors are aware that their newscasts scare the audiences they serve. Research suggests that local television news stations can improve ratings by reducing crime coverage and by changing the nature of coverage. Suggestions also are offered as to how audiences can be better prepared to react to biased reporting.

A survey measuring audience reactions to local television news was administered to middle school/high school students, college students, and senior citizens. The majority in each population reported watching the news to learn about the world. They agreed that news is mostly about violent events and that such news causes them to worry and to feel that the world is not a safe place. Significant sex differences were found indicating that women were more likely to report turning the news off and worrying about news content, whereas college men were more likely to watch to see coverage of violent events. The possibility of modifying news content was raised.

Curli are functional amyloid fibers assembled by many Gram-negative bacteria as part of an extracellular matrix that encapsulates the bacteria within a biofilm. A multicomponent secretion system ensures the safe transport of the aggregation-prone curli subunits across the periplasm and outer membrane, and coordinates subunit self-assembly into surface-attached fibers. To avoid the build-up of potentially toxic intracellular protein aggregates, the timing and location of the interactions of the different curli proteins are of paramount importance. Here we review the structural and molecular biology of curli biogenesis, with a focus on the recent breakthroughs in our understanding of subunit chaperoning and secretion. The mechanistic insight into the curli assembly pathway will provide tools for new biotechnological applications and inform the design of targeted inhibitors of amyloid polymerization and biofilm formation.

Patents on human gene sequences and relationships between gene variants and clinical phenotypes are controversial in clinical diagnostics. Proponents of gene patents claim they promote gene discovery and encourage the production of novel diagnostic tests. Opponents argue that they raise costs, decrease patient access, harm innovation in testing, and are unnecessary to promote gene discovery. The advent of large-scale sequencing has generated concerns that gene patenting will inhibit the implementation of new sequencing technologies. In the two US Supreme Court cases, Mayo v. Prometheus and Association for Molecular Pathology v. Myriad Genetics, the Court held that human DNA sequences and associations between biomarkers and human physiologic behavior represent unpatentable natural laws. These cases appear to have eliminated patent-based monopolization of clinical testing for mutations in human genes and genotype–phenotype relationships, and will help facilitate the introduction of next-generation sequencing into clinical practice. The incorporation of inherited disease testing into medical practice has generated apprehension about possibilities for “genetic discrimination” in employment and insurance. The Genetic Information Nondiscrimination Act (GINA) proscribes genetic discrimination in employment and health insurance. The exclusion of life, disability, and long-term care insurance from these protections has significant implications for reporting large-scale gene sequencing results.

Chronic pain is a major health problem, affecting 2%–46.5% of adult populations (1)(2). In the United States, common pain conditions account for approximately $61.2 billion in lost workplace time (3). The human sensation of pain is affected by an individual’s past experiences, health status, psychological state, cultural and ethnic backgrounds, pain-coping skills, age, sex, and even pending litigation (4)(5). Emotional variables profoundly influence the perception of painful stimuli (6). In addition, genetic contributions to the experience of pain have increasingly been recognized (7). However, the complexity of pain processing, the multiplicity of influences on it, and the sensation’s inherent subjectivity and protean manifestations make the elucidation of relevant heritable factors extremely challenging. Neuropathic pain is caused by injury to, or dysfunction of, the peripheral or central nervous system (8). It is particularly troublesome because of its prevalence, severity, chronicity, and resistance to therapy (9). Patients with conditions as diverse as diabetes mellitus, alcoholism, HIV, multiple sclerosis, postherpetic neuralgia, and spinal radiculopathies may suffer from chronic neuropathic pain. Radiculopathic pain is among the most frequently encountered neuropathic pain syndromes (8)(9). Sciatica associated with intervertebral disc herniation is the most common type of radicular leg pain in adult working populations (10). Although many patients with herniated discs have favorable outcomes with medical therapy, individuals who have ongoing or severe pain often undergo lumbar discectomy. Recently, Tegeder et al. (11) described a putative association between an apparently common haplotype (15.4% of alleles in the study population) of the GTP cyclohydrolase gene (dopa-responsive dystonia, GCH1 ) and lower degrees of persistent pain after back surgery. GTP cyclohydrolase is the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis. BH4 serves as an essential cofactor in the production of catecholamines, serotonin, and nitric oxide, as well as in phenylalanine metabolism. Inactivating mutations …

Overview: The use of genetic information to design and guide therapies and to develop novel diagnostic procedures creates important patent issues. Patents on human gene sequences have likely helped stimulate the introduction of new biologics; however, their role and that of patents on genotype-phenotype correlations in diagnostic testing is highly controversial. Genotype-phenotype associations are at the heart of personalized medicine. The intellectual property rules by which these biologic relationships are governed have profound implications for the growth of individualized medicine.

Legal and regulatory issues have assumed heightened importance in molecular pathology. Patents on relationships between human gene variants and clinical phenotypes, as well as on the underlying gene sequences themselves, have been extremely controversial. However, recent Supreme Court decisions appear to have rendered invalid these classes of patents. The Clinical Laboratory Improvement Amendments of 1988 have been the primary basis for laboratory oversight in the United States, whereas the Food and Drug Administration has been responsible for regulating in vitro test kits distributed in interstate commerce. However, the Food and Drug Administration has recently announced its intention to regulate laboratory-developed tests, raising concerns in the laboratory community. This article reviews recent developments impacting the legal status of gene patenting and oversight of molecular pathology testing in the United States.

With the accelerated implementation of genomic medicine, health-care providers will depend heavily on professional guidelines and recommendations. Because genomics affects many diseases across the life span, no single professional group covers the entirety of this rapidly developing field. To pursue a discussion of the minimal elements needed to develop evidence-based guidelines in genomics, the Centers for Disease Control and Prevention and the National Cancer Institute jointly held a workshop to engage representatives from 35 organizations with interest in genomics (13 of which make recommendations). The workshop explored methods used in evidence synthesis and guideline development and initiated a dialogue to compare these methods and to assess whether they are consistent with the Institute of Medicine report “Clinical Practice Guidelines We Can Trust.” The participating organizations that develop guidelines or recommendations all had policies to manage guideline development and group membership, and processes to address conflicts of interests. However, there was wide variation in the reliance on external reviews, regular updating of recommendations, and use of systematic reviews to assess the strength of scientific evidence. Ongoing efforts are required to establish criteria for guideline development in genomic medicine as proposed by the Institute of Medicine. Genet Med advance online publication 19 June 2014

The Evaluation of Genomic Applications in Practice and Prevention Working Group was first convened in 2005 to develop and test evidence-based methods for the evaluation of genomic tests in transition from research to clinical and public health practice. Over the ensuing years, the Working Group has met 26 times, publishing eight recommendation statements, two methods papers, and one outcomes paper, as well as planning and serving as technical experts on numerous associated systematic reviews. Evaluation of Genomic Applications in Practice and Prevention methods have evolved to address implications of the proliferation of genome-wide association studies and are currently expanding to face challenges expected from clinical implementation of whole-genome sequencing tests. In this article, we review the work of the Evaluation of Genomic Applications in Practice and Prevention Working Group over the first 8 years of its existence with an emphasis on lessons learned throughout the process. It is hoped that in addition to the published methods of the Working Group, the lessons we have learned along the way will be informative to others who are producers and consumers of evidence-based guidelines in the field of genomic medicine. © American College of Medical Genetics and Genomics.

The guest editorial provides perspective to the decision by the Supreme Court of the United States in the case of Association for Molecular Pathology v Myriad Genetics, Inc.

Small heat shock proteins (sHsps) are molecular chaperones that protect cells from the effect of heat and other stresses. Some sHsps are also expressed at specific stages of development. In plants different classes of sHsps are expressed in the various cellular compartments. While the Class I (cytosolic) sHsps in wheat and pea have been studied extensively, there are fewer experimental data on Class II (cytosolic) sHsps, especially in maize. Here we report the expression and purification of two Class II sHsps from Zea mays ssp. mays L. (cv. Oh43). The two proteins have almost identical sequences, with the significant exception of an additional nine-amino-acid intervening sequence near the beginning of the N-terminus in one of them. Both ZmHsp17.0-CII and ZmHsp17.8-CII oligomerize to form dodecamers at temperatures below heat shock, and we were able to visualize these dodecamers with TEM. There are significant differences between the two sHsps during heat shock at 43°C: ZmHsp17.8-CII dissociates into smaller oligomers than ZmHsp17.0-CII, and ZmHsp17.8-CII is a more efficient chaperone with target protein citrate synthase. Together with the previous observation that ZmHsp17.0-CII but not ZmHsp17.8-CII is expressed during development, we propose different roles in the cell for these two sHsps.

Since 2006, the US Food and Drug Administration, Congress, and other policymakers have explored the appropriate way to guarantee the clinical and analytical validity of laboratory-developed tests. In the past, the Association for Molecular Pathology has publicly urged the Food and Drug Administration to exercise caution in implementing regulatory changes that could potentially hinder innovation or interfere with the practice of medicine. In 2012, the Association for Molecular Pathology Professional Relations Committee chose to develop this paper with the goal of outlining the best methods for ensuring appropriate oversight and validation of molecular diagnostic procedures. At the conclusion of this process, the workgroup reaffirmed the Association's previous position that the Centers for Medicare and Medicaid Services Clinical Laboratory Improvement Amendments program can provide the appropriate level of oversight for the vast majority of diagnostic tests.

The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (EWG) found that, for patients with metastatic colorectal cancer (mCRC) who are being considered for treatment with cetuximab or panitumumab, there is convincing evidence to recommend clinical use of KRAS mutation analysis to determine which patients are KRAS mutation positive and therefore unlikely to benefit from these agents before initiation of therapy. The level of certainty of the evidence was deemed high, and the magnitude of net health benefit from avoiding potentially ineffective and harmful treatment, along with promoting more immediate access to what could be the next most effective treatment, is at least moderate.The EWG found insufficient evidence to recommend for or against BRAF V600E testing for the same clinical scenario. The level of certainty for BRAF V600E testing to guide antiepidermal growth factor receptor (EGFR) therapy was deemed low. The EWG encourages further studies of the potential value of testing in patients with mCRC who were found to have tumors that are wild type (mutation negative) for KRAS to predict responsiveness to therapy.The EWG found insufficient evidence to recommend for or against testing for mutations in NRAS, or PIK3CA, and/or loss of expression of PTEN or AKT proteins. The level of certainty for this evidence was low. In the absence of supporting evidence, and with consideration of other contextual issues, the EWG discourages the use of these tests in guiding decisions on initiating anti-EGFR therapy with cetuximab or panitumumab unless further evidence supports improved clinical outcomes.It has been suggested that patients with mCRC whose tumors harbor certain mutations affecting EGFR pathway signaling are typically unresponsive to therapy with anti-EGFR antibodies (cetuximab and panitumumab). The EWG identified recent evidence reviews that have addressed this topic, and this recommendation statement is based on results of these reviews. In developing these recommendations the EWG considered evidence in the areas described below.Although no research syntheses that have formally evaluated analytic validity of these tests were found, the EWG was able to draw the following conclusions from assessments included in the evidence reviews under consideration. There is adequate evidence that KRAS mutation analysis reliably and accurately detects common mutations (codons 12 and 13), whereas evidence was inadequate for less frequent KRAS mutations (e.g., codon 61). There is also adequate evidence that testing for BRAF V600E accurately and reliably detects the mutation. For common mutations in NRAS, PIK3CA, and expression of PTEN AKT, there is adequate evidence of accurate and reliable detection. However, much less data exist in support. Furthermore, in the specific context of mCRC, no evidence was found on the analytic validity of immunohistochemistry (IHC) assays for PTEN or AKT expression.For KRAS mutation analysis, the EWG found convincing evidence for association with treatment response to anti-EGFR therapy, independent of prognostic association. For BRAF V600E mutation testing, the EWG found insufficient evidence for association with treatment response to anti-EGFR therapy independent of prognostic association. The EWG found insufficient evidence for association of results of testing for mutations in NRAS or PIK3CA, and loss of expression of PTEN or ATK proteins, with treatment response to anti-EGFR therapy.For KRAS mutation analysis, the EWG found adequate evidence that improved health outcomes are achieved by avoiding ineffective chemotherapy and potential side effects and expediting access to the next most effective treatment. Inadequate evidence was found regarding association of BRAF V600E mutation testing or loss of PTEN expression with improved health outcomes among patients with mCRC undergoing anti-EGFR therapy as compared with patients with tumors bearing wild-type BRAF sequence and PTEN expression levels, respectively. No evidence was found to support improved health outcomes associated with testing results for NRAS or PIK3CA variants, or AKT protein expression levels in this clinical scenario.CRC is an important and highly prevalent health problem. Improvements in mCRC outcomes associated with pharmacogenetic testing could have important clinical, and potentially public health, impacts. Adverse events related to cancer chemotherapy can be common and severe. Therefore, successfully optimizing treatment to maximize efficacy and minimize side effects is important for reducing mCRC-related morbidity and mortality.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.